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Synthesis of Anti-angiogenetic Natural-like Acylphloroglucinols
and
Selective ABC Transporter Modulators
Dissertation
Zur Erlangung des Doktorgrades der Naturwissenschaften
(Dr. rer. nat.)
der Fakultät für Chemie und Pharmazie
der Universität Regensburg
vorgelegt von
Qiu Sun
aus
Chengdu (China)
2015
The experimental part of this work was carried out between October 2011 and December 2014
under the supervision of Prof. Dr. Burkhard König at the Institute of Organic Chemistry,
University of Regensburg.
The thesis was submitted on: 2nd, March, 2015
Date of the colloquium: 27th, March, 2015
Board of examiners: Prof. Dr. Achim Göpferich (Chairman)
Prof. Dr. Burkhard König (1st Referee)
Prof. Dr. Jörg Heilmann (2nd Referee)
PD Dr. Sabine Amslinger (Examiner)
Dedicated
To
My family
路漫漫其修远兮，吾将上下而求索。
The journey is long; I'll search up and down.
屈 原
Qu Yuan
Table of Contents
Chapter 1 ...................................................................................................................................................................... 1
Natural phenolic metabolites with anti-angiogenic properties – a review from the chemical point of view ....... 1
Abstract ..................................................................................................................................................................... 1
Introduction ............................................................................................................................................................... 3
4-Hydroxybenzyl alcohol.......................................................................................................................................... 8
Curcumin .................................................................................................................................................................. 9
Ellagic acid ............................................................................................................................................................. 12
Resveratrol .............................................................................................................................................................. 13
Quinoline substituted phenols ................................................................................................................................. 14
4-Amino-2-sulfanylphenol derivatives ................................................................................................................... 16
Acylphloroglucinol derivatives ............................................................................................................................... 17
(-)-Epigallocatechin-3-O-gallate (EGCG) .............................................................................................................. 20
Xanthohumol .......................................................................................................................................................... 22
Genistein ................................................................................................................................................................. 24
Fisetin and Quercetin .............................................................................................................................................. 24
(2S)-7,2’,4’-Trihydroxy-5-methoxy-8-dimethylallyl flavanone ............................................................................. 27
Conclusions ............................................................................................................................................................. 27
References ............................................................................................................................................................... 28
Chapter 2 .................................................................................................................................................................... 33
Synthesis of natural and natural-like acylphloroglucinols with anti-proliferative, anti-oxidative and tube-
formation inhibitory activity .................................................................................................................................... 33
Abstract ................................................................................................................................................................... 33
Introduction ............................................................................................................................................................. 35
Results and discussion ............................................................................................................................................ 36
Conclusions ............................................................................................................................................................. 42
Experimental ........................................................................................................................................................... 43
References ............................................................................................................................................................... 53 1H and
13C NMR spectra of selected final compounds ........................................................................................... 55
Chapter 3 .................................................................................................................................................................... 58
Flavonoid derivatives as selective ABCC1 modulators: synthesis and functional characterization .................. 58
Abstract ................................................................................................................................................................... 58
Introduction ............................................................................................................................................................. 59
Results and discussion ............................................................................................................................................ 60
Conclusions ............................................................................................................................................................. 72
Experimental ........................................................................................................................................................... 72
References ............................................................................................................................................................... 94
1H and
13C NMR spectra of selected final compounds ........................................................................................... 99
Chapter 4 .................................................................................................................................................................. 102
Quinoline carboxamide-type ABCG2 modulators: quinoline moiety as anilide replacement .......................... 102
................................................................................................................................................................................... 102
Abstract ................................................................................................................................................................. 102
Introduction ........................................................................................................................................................... 103
Results and Discussion ......................................................................................................................................... 104
Conclusions ........................................................................................................................................................... 112
Experimental ......................................................................................................................................................... 112
References ............................................................................................................................................................. 130 1H and
13C NMR spectra of selected final compounds ......................................................................................... 132
Chapter 5 .................................................................................................................................................................. 135
Triphenylphosphine mediated photo-rearrangement and methanol addition of aryl chalcones to 1-
propanones ............................................................................................................................................................... 135
Abstract ................................................................................................................................................................. 135
Introduction ........................................................................................................................................................... 136
Results and Discussion ......................................................................................................................................... 137
Conclusions ........................................................................................................................................................... 143
Experimental ......................................................................................................................................................... 143
References ............................................................................................................................................................. 151 1H and
13C NMR spectra of selected compounds ................................................................................................. 154
Abbreviation ............................................................................................................................................................ 157
Summary .................................................................................................................................................................. 159
Zusammenfassung ................................................................................................................................................... 161
Curriculum Vitae ..................................................................................................................................................... 163
Acknowledgement .................................................................................................................................................... 167
Chapter 1 | 1
This chapter has been published:
Q. Sun, J. Heilmann and B. König. Beilstein J. Org. Chem. 2015, 11, 249-264.
Author contributions:
Q. Sun wrote the manuscript.
Chapter 1
Natural phenolic metabolites with anti-angiogenic properties – a review from
the chemical point of view
Abstract
Within the secondary natural metabolites from plants, phenolic compounds have a special impact
on human health as they occur in significant amounts in several fruits, vegetables and medicinal
plants. In this review natural phenolic compounds of plant origin with significant anti-angiogenic
properties are summarized. Thirteen representatives of eight different natural or natural like
Chapter 1 | 2
phenolic subclasses are presented with a particular emphasis on their synthesis and the methods
to modify the parent compounds. Whenever available, the consequence of structural variation on
the pharmacological activity of the molecules is described.
Keywords
natural phenolic compounds, angiogenesis, synthesis, structure-activity relationship
Chapter 1 | 3
Introduction
The term “angiogenesis” is commonly used to describe the biological process of blood vessel
growth. Nevertheless, it should be more precisely defined as formation of new blood vessels
from pre-existing ones. Under physiological conditions angiogenesis is vital for foetal
development, tissue regeneration and wound healing. Patho-physiologically, massive vascular
growth or abnormal shape formation promotes many diseases including cancer, inflammation,
and eye illness. However, inadequate vessel preservation or growth leads to ischemia causing
myocardial infarction, stroke, and neurodegenerative or obesity-associated conditions.[1]
The generation of new blood vessels is based on a strictly controlled balance between various
soluble and membrane-bound factors showing either anti- or pro-angiogenic activity and thus
embedded together with enzymes and signalling molecules (Table 1) into a very complex
network of signal pathways.[2]
In case of cancer development the growing tumor disturbs the
angiogenic balance in a tissue and induces the secretion of pro-angiogenic factors either by the
tumor cells or by cells of the tumor microenvironment. When the tumor grows to the diameter of
1-2 mm, the tumor cells located far away from blood vessels undergo apoptosis or necrosis
resulting from the lack of oxygen and nutrients. At this moment, tumor cells express pro-
angiogenic factors including growth factors such as the vascular endothelial growth factor
(VEGF) and fibroblast growth factor (FGF) and enzymes like cyclo-oxygenase 2 (COX-2) and
protein kinase A (PKA) as well as signalling molecules like the integrins. The evoked cascades
induce the formation of new blood vessels quickly connected with the pre-existing blood vessels
providing sufficient supplies for tumor survival.[3]
In addition, the new blood vessels allow
cancer cells to transfer from a parent location to other new locations causing metastases.
Nevertheless, the morphology and pathophysiology of these blood vessels differs significantly
from physiological ones as they work less effective and show a lower state of organization and
control.[2a]
After discovering the mechanism of angiogenesis and its crucial role in the tumor
development, different therapies targeting to interfere with this process were investigated.[4]
Preferred clinical target are the VEGF receptors leading to the development and approval of
monoclonal antibodies against VEGF and VEGF receptor tyrosine kinase inhibitors.[2a]
Nevertheless, the existing therapy options with antibodies and VEGF receptor inhibitors showed,
Chapter 1 | 4
from the clinical point of view, several limitations making the search for further clinically
relevant targets and other drugs mandatory to combat tumor related angiogenesis.
Table 1. Condensed overview on endogenous modulators involved in angiogenic processes
Group name Modulators (abbreviation)
Growth factors Vascular endothelial growth factor (VEGF)
Fibroblast growth factor (FGF)
Platelet-derived growth factor (PDGF)
Transforming growth factor β1 (TGF-β1)
Angiopoetins (Ang)
Adhesion molecules Integrins
Cadherins
Proteinases Matrix metalloproteinases (MMPs)
Urokinase plasminogen activator (uPA)
Extracellular matrix proteins Fibronectin
Collagens
Transcription factors Nuclear factor (erythroid-derived 2)-related factor (Nrf2)
Nuclear factor 'kappa-light-chain-enhancer' of activated B-
cells (NF-κB)
Activator protein (AP-1)
Hypoxia inducible factor (HIF)
Other signalling molecules Mammalian target of rapamycin (mTOR)
Other enzymes Mitogen-activated-protein-kinases (MAP-kinases)
Proteinkinases A and C (PKA and PKC)
Proteinkinase B/Akt
Cyclo-oxygenase 2 (COX-2)
Nitric oxide synthase (NOS)
Chapter 1 | 5
Accumulating research also showed that secondary natural metabolites are attractive candidates
for the therapy of pathologically induced angiogenesis.[5]
Among such natural products phenolic
or polyphenolic compounds with anti-angiogenic properties have been investigated and the
opinion on their pharmacological impact has changed over the last years. While the
pharmacological activity of polyphenols was previously considered as unspecific, more recently
observations of a specific interference with biological mechanisms at the molecular level are
exponentially growing. Especially in the field of anti-inflammatory activity, chemoprevention
and cytoprotection natural phenolic metabolites like flavonoids, caffeic acid derivatives and
diarylheptanoids showed pleiotropic influence on cellular signalling e. g. by the inhibition of
transcription factors like NF-B or Nrf2,[6]
or anti-oxidative effects.[6b, 7]
Furthermore,
polyphenols are abundant in many plants used as fruits and vegetables in high concentrations,
resulting in a continuous and long-term intake of such plant phenols. Consequently, their
beneficial and protective impact on unbalanced angiogenic processes has been intensively
discussed.[5]
In the last decade, many excellent review articles summarized the biological and
pharmacological aspects of anti-angiogenic compounds including natural compounds with a
phenolic substructure.[8]
Complementary to the previously discussed pharmacological point of
view this review focuses on recent reports of anti-angiogenic natural phenolic compounds
specifically addressing their chemistry, synthesis and possible structure modifications.
Nevertheless, it should be mentioned that the selection of compounds for this review is based on
the reports on their pharmacological activity. As the term “anti-angiogenic compound” is not
unequivocally defined and somewhat inflationarily used, appropriate inclusion criteria for the
review had to be defined. Compounds included in our survey have shown anti-angiogenic
activity not only in convenient (and often descriptive) cellular in vitro assays (Table 2), but also
in molecular in vitro test systems related to the signalling cascades of pathological angiogenesis.
Further inclusion criteria were the existence of anti-agiogenic activity obtained in ex vivo and in
vivo assays (Table 2). Additionally, the observed in vitro activity should be in the lower µM
range (or better) and thus high enough to realistically speculate on an anti-angiogenic activity in
vivo. As endothelial cells (ECs) have an extraordinary significance in angiogenesis, results from
cellular assays using primary or immortalized ECs like human umbilical vein endothelial cells
(HUCEC) or human microvascular endothelial cells (HMEC-1) have been given special attention.
Chapter 1 | 6
In contrast, compounds showing in vitro anti-angiogenic activity, but also most likely signs of
strong unspecific cytotoxic effects in vitro have been excluded. The discussed secondary
metabolites include six flavonoids from different subclasses namely quercetin, fisetin,
epigallocatechin-3-O-gallate, xanthohumol, (2S)-7,2’,4’-triihydroxy-5-methoxy-8-dimethylallyl
flavanone and genistein. Other compounds belong to the groups of simple phenols (4-
hydroxybenzyl alcohol), hydrolysable tannins (ellagic acid), stilbenoids (resveratrol) and
diarylheptanoids (curcumin). In addition, acylphloroglucinols, quinoline substituted phenols and
4-amino-2-sulfanylphenol derivatives were discussed. Some important aspects of the described
pharmacological activities of the compounds are summarized in Table 3.
Table 2. In vitro, ex vivo and in vivo assays to characterize anti-angiogenic activity*
In vitro assays Assay principles / detection, read out
Endothelial cell proliferation
assays
Cell counting / Increase of cell number
Crystal violet / Increase of cell number
MTT / Activity of dehydrogenase activity (positively
correlated to cell number)
Incorporation of [3H]thymidine, 5-bromodeoxyuridine into
DNA / DNA synthesis (positively correlated to cell number)
Endothelial cell migration
assays
Scratch assay / Migration into a denuded area (wound
healing)
Endothelial cell differentiation
assays
Tube formation e.g. in Matrigel/ Formation of capillary like
tubules
Endothelial-Mural cell co-
culture assays
Interaction between two cell types (endothelial/mural) /
Influence on cell differentiation and proliferation
Ex vivo assays
Aortic ring assay Aorta of rodents cultured in biological matrices / Outgrowth
of branching microvessels
In vivo assays
Chapter 1 | 7
-Table 2 continued-
Chick chorioallantoic membrane
assay (CAM)
Extra-embryonic membrane (in ovo, ex ovo) / growth and
branching of blood vessels
Hen's egg test on chorioallantoic
membrane (HETCAM)
CAM modification / Growth and branching of blood vessels
Zebrafish Zebrafish embryos or transgenic zebrafish embryos /
Visualization of vascularisation (e.g. with confocal
microscopy)
Corneal angiogenesis assay Corneal injury or implantation of pellets / Vascular response
of the cornea
Dorsal air sac model Ring (filled with tumor cell suspension) implantation (dorsal
skin) / Tumor induced angiogenesis
Mouse models Genetic engineered mouse models; xenografts
*Molecular or enzyme assays not included
Table 3. Natural phenolic compounds with anti-angiogenic activity and their evaluated molecular
mechanisms of anti-angiogenesis
Compound name Mechanisms of anti-angiogenic action
4-Hydroxybenzyl alcohol Down-regulation of VEGF and MMP9 protein expression
Curcumin Reduction of VEGF expression, inhibition of transcription
factors, mTOR pathway and MMP9 protein expression
Ellagic acid Inhibition of VEGF and PDGF receptor phosphorylation
Resveratrol Abrogation of VEGF-mediated tyrosine phosphorylation of
vascular endothelial (VE)-cadherin, inhibition of VEGF-
induced and FGF-2 neovascularization
Quinoline substituted phenols Inhibition of VEGF and transforming growth factor-β1 (TGF-
β1) expression
Chapter 1 | 8
-Table 3 continued-
4-Amino-2-sulfanylphenol
derivatives
Inhibition of protein kinase B/Akt and ABL tyrosine kinase
Nature-like acylphloroglucinol
derivatives
Under investigation
(-)-Epigallocatechin gallate
(EGCG)
Inhibition of estrogen-stimulated VEGF expression, HIF-1α
and Nf-κB, inhibition of MMP-2 and MMP-9, inhibition of
urokinase plasminogen activator
Xanthohumol Inhibition of Nf-κB and Akt pathways
Genistein Inhibition of VEGF and HIF-1α protein expression
Fisetin Down-regulation of VEGF and eNOS expression, inhibition
of MMPs activities
Quercetin
Inhibition of the expression of VEGF-2, inhibition of COX-2
and arachidonate 5-lipoxygenase (LOX-5), inhibition of Nf-
B, In some cell types it activates angiogenesis.
(2S)-7,2’,4’-Triihydroxy-5-
methoxy-8-dimethylallyl
flavanone
Down-regulation of reactive oxygen speics (ROS) levels and
VEGF expression
4-Hydroxybenzyl alcohol
4-Hydroxybenzyl alcohol (HBA, 1) (Figure 1) is a well-known phenolic compound from plants
and has been for example found in flowers of carrot (Daucus carrota L., Apiaceae). In 2007,
Park and co-workers [9]
found in the chick chorioallantoic membrane (CAM) assay no change of
the vascular density in the presence of HBA, indicating that HBA has no influence on the growth
of blood vessels. In contrast, the branching pattern of blood vessels was reduced dose-
dependently in the same assay, making an inhibition of angiogenesis likely. Later, Laschke et al.
(2011) [10]
performed experiments in vitro with an aortic ring assay and in vivo in an
Chapter 1 | 9
endometriosis model as well as systematic analysis of the mechanism underlying the anti-
angiogenic activity of HBA. They found that HBA is capable of inhibiting several steps of the
angiogenic mechanism. Western blot analysis showed the down-regulation of VEGF and MMP9
protein expression. The effect of HBA was confirmed [11]
by mouse dorsal skinfold chamber
experiments. Incubation of CT26.WT colon carcinoma cells with HBA showed a dose dependent
decrease of their viability and integrity. In addition, the cells expression of the apoptosis marker
cleaved caspase-3 increased significantly and the expression of vascular endothelial growth
factor (VEGF) and matrix metalloproteinase (MMP)-9 decreased compared to controls. No
influence on the normal behaviour of the animals was observed. In general, HBA represents an
interesting anti-angiogenic agent for the treatment of angiogenic diseases.
Figure 1. Structure of 4-hydroxybenzyl alcohol (HBA).
Curcumin
Curcumin (3) is a natural product isolated from different Curcuma species (Zingiberaceae) some
of them used as raw material of the spice turmeric. It has been evaluated as a chemopreventive
agent since the early nineties and in 1998, Arbiser and his co-workers found that the compound
showed also anti-angiogenic properties in vitro and in vivo.[12]
In the following years, many
studies on the anti-angiogenic properties in different tumor cell lines or in animal models were
reported.[13]
They include interactions with the transcription factor Nf-B, mTOR pathway, and
reduction of VEGFA and MMP9 expression. Despite of its promising pharmacological
properties, curcumin suffers from a low in vivo bioavailability as a consequence of its low
aqueous solubility, poor chemical stability and low adsorption. Therefore many analogues
(Figure 2) were synthesised in order to overcome these drawbacks and enhance the activity. In
addition, their structure-activity relationships were studied to gain better insight into the mode of
action. The general synthesis of curcumin itself (Scheme 1) requires masking of the reactive
Chapter 1 | 10
methylene group of acetylacetone by formation of a complex with boric oxide, followed by
reaction with vanillin. Instead of boric oxide, alkyl borate esters and boric acid can be used.[14]
The first attempt of modification was to truncate the general structure to either a single enone or
dienone system. The latter group showed a diarylpentanoid instead of the natural diarylheptanoid
backbone, in some cases amended by a central ring system, and was labelled as monocarbonyl
analogues of curcumin (MACs, Figure 3). Bowen et al.(2003) [15]
used Claisen–Schmidt reaction
for synthesis of these analogues. The C7- chain between the two aromatic rings was shortened
and a series of compounds (Scheme 2) with different substitutions on the aromatic rings was
synthesised to explore stereoelectronic effects. It was demonstrated that those analogues of
curcumin were excellent anti-angiogenic compounds, having inhibition patterns equivalent or
better than the parent natural product. This work was continued by more comprehensive
bioactive studies on aromatic enones utilizing the substituted chalcone backbone.[16]
The study
showed that the presence of the enone moiety played an important role in maintaining the
activity in the curcumin analogues. Ahn et al. (2005) [17]
left the enone part unchanged to the
previous principle and prepared various curcumin mimics with asymmetric units with bearing
alkyl amide, chloro-substituted benzamide, or heteroaromatic amide moieties. Those analogues
showed stronger anti-angiogenic activity than curcumin against HUVEC. Up to now the number
of synthesised single enones and MACs clearly broke the 1000 mark.
Scheme 1: Synthesis of Curcumin 3. Reagents and conditions: (a) vanillin, 1,2,3,4-tetrahydroquinolin,
HOAc, H3BO3, DMF, Δ 4 h.
Chapter 1 | 11
Figure 2. Structure-activity relationship of curcumin analogues.
Figure 3. Backbone and substitution of monocarbonyl analogues of curcumin (MACs) showing their
structural diversity.
Chapter 1 | 12
Scheme 2. Exemplary synthesis of monocarbonyl analogues of curcumin (MACs). Reagents and
conditions: (a) 40% KOH, EtOH, 5°C, stir 10 h, rt. X=C, N. R=OH, OMe, Cl, F.
Ellagic acid
Ellagic acid (7) is a naturally existing phenol antioxidant widely found in numerous fruits and
vegetables like raspberries (Rubus idaeus L., Rosaceae), strawberries (Fragaria spec. L.,
Rosaceae) and pomegranates (Punica granatum L., Lythraceae). It shows potent antioxidant
effects by radical scavenging and the inhibition of lipid peroxidation.[18]
Ellagic acid is also
capable of interfering with some angiogenesis-dependent pathways. It possesses anti-
carcinogenic activity through inhibiting tumor cell proliferation, migration and induction of
apoptosis. In addition, it is a dual inhibitor of the phosphorylation of VEGF and PDGF receptors,
intercepting the angiogenesis processes required for tumor growth.[19]
Recently, it was reported
that its anti-angiogenesis mechanism affects the VEGFR-2 signalling pathway by forming
hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR-2
kinase unit.[20]
Shankar and Srivastava et al. (2013) [21]
treated PANC-1 xenografted mice with
the ellagic acid and measured the expression of Akt, Shh and Notch. The results suggested that
ellagic acid effectively inhibited human pancreatic cancer growth by suppressing protein kinase
B (Akt), sonic hedgehog (Shh) and Notch pat hways. The preparation of ellagic acid (7) can be
achieved by oxidative coupling of gallic acid (Scheme 3).[22]
In the presence of H2SO4, gallic
acid (4) was esterified to methyl gallate (5). The gallate (5) was oxidized by o-chloranil followed
by reduction with Na2S2O4 to obtain the hexahydroxy biphenyl (6). Subsequent lactonization
afforded the final product ellagic acid (7) in high yield.
Chapter 1 | 13
Scheme 3. Synthesis of ellagic acid 7. Reagents and conditions: (a) H2SO4, CH3OH; (b) (1) o-chloranil,
Et2O, -40°C; -40°C → r.t, 3 h; (2) Na2S2O4, r.t, 30 min; (c) MeOH:H2O=1:1, reflux.
Resveratrol
Resveratrol (8) is a natural polyphenol belonging to the stilbenoids and widely existing in a
number of plants. It was primarily extracted from grape (Vitis vinifera L., Vitaceae), and
mulberry (Morus L., Moraceae) (Figure 4).[23]
It has anti-oxidant effects, anti-estrogenic
activities and the ability to reduce the synthesis of hepatic lipids and eicosanoids. It inhibits
platelet aggregation and protects vessels from arteriosclerosis.[23-24]
In recent years, it has been
reported that resveratrol is sufficiently potent to inhibit VEGF-induced and FGF-2
neovascularization in vivo.[25]
It was also found that resveratrol showed direct inhibition to
bovine aorta endothelial cell proliferation, migration and tube formation in vitro.[26]
Resveratrol
has also been found to effectively abrogate VEGF-mediated tyrosine phosphorylation of vascular
endothelial (VE)-cadherin and its complex partner, β-catenin.[27]
But unfortunately, resveratrol
has dual effects on cells depending on the situation and cell type, meaning it can either induce or
suppress angiogenic effects.[28]
The low oral bioavailability and metabolic stability of resveratrol
also limited its application.[29]
Therefore, in an attempt to increase its bioavailability and stability,
Chapter 1 | 14
the structure of resveratrol was modified by methylation of the phenol group [30]
and introduction
of other groups on the phenyl ring (see compound 9-15).[31]
Trans-3,4,5,4’-tetramethoxystilbene
(DMU-212) has pharmacokinetic properties that are better compared to resveratrol and shows
anti-proliferative activities in different cancer cells.[32]
The further investigation of its role in
angiogenesis by Dai and Zhang et al.(2013) [33]
showed that DMU-212, a potential anti-
angiogenic agent, inhibits VEGFR2 phosphorylation and thereby acts as a suppressor of
signalling pathways mediated by VEGFR2 inducing apoptosis in endothelial cells.
Figure 4. Structure of resveratrol 8 and its analogues.
Quinoline substituted phenols
Up to now, quinoline substituted phenols (Qsps) have not been reported as natural secondary
metabolites, but the individual substructures quinoline and alkyl phenol are common structural
elements of secondary plant products. The quinoline skeleton is present in alkaloids derived from
tryptophane, like quinine or camptothecine, whereas alkyl phenols with a varying length of the
alkyl side chain are common metabolites from the shikimate pathway. Qsps were reported in a
recent patent to be effective for the treatment of angiogenesis-related diseases or disorder.
Among other assays a transgenic line of zebrafish that express a fluorescent reporter (EGFP) in
vasculature was used in their study to identify anti-angiogenic compounds.[34]
They particularly
Chapter 1 | 15
looked at the integrity of vessels developing in the eyes and in the trunk. Quinoline-substituted
phenols were identified being active based on a significant inhibition of the hyaloid vessel
formation in the zebrafish model. The synthesis of two representative compounds 20 and 23 of
this class are shown in Scheme 4 and Scheme 5.[34]
Scheme 4. Synthesis of quinoline substituted phenol 20. Reagents and conditions: (a) Ac2O, 2-
hydroxybenzaldehyde, 130°C; (b) (1) Br2, AcOH; (2) Ac2O; (c) (1) DBU, THF; (2) Ac2O; (d) Na2CO3,
MeOH/THF.
Scheme 5. Synthesis of quinoline substituted phenol 23. Reagents and conditions: (a) Ac2O, 2-
hydroxybenzaldehyde; (b) (1) NaOH, EtOH/H2O, 100°C; (2) HCl.
After condensation of 16 with an aldehyde, product 17 was subsequently brominated, followed
by twofold elimination of HBr forming a triple bond. Finally, ester hydrolysis provides
Chapter 1 | 16
compound 20. Compound 23 was obtained from an analogous condensation product 22
hydrolysis under basic condition.
4-Amino-2-sulfanylphenol derivatives
The group of 4-amino-2-sulfanylphenols is obviously an outlier among the reviewed compounds
as the 2-sulfanylphenol structure is not a structural element in natural products. The vast majority
of thiol groups in secondary metabolites derive from the amino acid from cysteine, producing
aliphatic secondary metabolites with thiol functionality instead of phenolic ones. Nevertheless,
this group contains thiol-analogues of the naturally occurring o-catechol substructure and thus
they have been included to this review. Zhang and Xu et al. (2013) [35]
have reported that 4-
amino-2-sulfanylphenol compounds display high specific protein kinase and angiogenesis
inhibitory activities. Based on their previous findings, the structure of compound 24 was
optimized by replacing the naphthalene ring by a phenolic skeleton and a sulfonamide
fragment.[36]
These compounds show in vitro anti-angiogenic activities compared to Pazopanib in
both human umbilical vein endothelial cell (HUVEC) tube formation assay and the rat thoracic
aorta rings test. They inhibited protein kinase B/Akt and ABL tyrosine kinase in the micro-molar
range. The preliminary structure-activity relationship is summarized in Figure 5.
Figure 5. Design of 4-amino-2-sulfanylphenol derivatives and their structure-activity relationship.
Chapter 1 | 17
The synthesis of 4-amino-2-sulfanylphenol compounds (Scheme 6) [37]
starts from the 4-
aminophenol hydrochloride salt 25, which was dissolved in pyridine and reacted with various
substituted sulfonyl chlorides yielding compounds 26 that differ in the substituent R1. Oxidation
by NaIO4/SiO2 gives the quinone-type structures 27, which were reacted without further
purification with thiols R2SH. The addition to the unsaturated system yielded the target
compounds 28 with different arylthiol groups under rearomatization.
Scheme 6. Synthesis of 4-amino-2-sulfanylphenol deriatives. Reagents and conditions: (a) R1SO2Cl,
Pyridine, 0°C; (b) NaIO4/SiO2, DCM; (c) DMF, R2SH.
Acylphloroglucinol derivatives
Acylphloroglucinols are typical secondary metabolites biosynthetically derived from the
polyketide pathway and mainly accumulating in Hypericaceae [38]
and Clusiaceae. Hyperforin,
likely the most prominent acylphloroglucinol derivative and present in higher amounts in St.
John’s wort (Hypericum perforatum L., Hypericaceae), has been recently reported to exhibit
strong anti-proliferative effects [39]
and strongly inhibited angiogenesis in vitro and in vivo
models. Mechanistically, it interferes with MMP-2 and an urokinase plasminogen activator
Chapter 1 | 18
(uPA),[40]
but due to its instability in aqueous solution, complex structure and limited availability,
hyperforin is neither a drug candidate nor a good model compound. The finding that structurally
simpler natural acylphloroglucinol derivatives showed also anti-proliferative effects against
endothelial cells with inhibitory effects in a tube-formation assay on Matrigel catalysed the
search for simple acylphloroglucinols with anti-angiogenic activity.[41]
Within this group some
geranylated monocyclic and bicyclic acylphloroglucinol derivatives have been found, which are
structurally much simpler than hyperforin, but exhibiting potent anti-proliferative activity for a
human microvascular endothelial cell line (HMEC-1) at low micromolar concentration.[41]
Two
series of natural-like acylphloroglucinols were synthesised (Figure 6) for the systematic
investigations of their anti-angiogenic properties.[42]
Compound 47 (R1=H, R2=OH, R3=OH)
showed anti-proliferative activity with an IC50 of 0.88 ± 0.08 µM in vitro. Compound 38 (alkyl =
CH(CH3)CH2CH3) exhibited moderate anti-proliferative effects (IC50 =11.0 ± 1 µM) and
inhibited the capillary-like tube formation of HMEC-1 in vitro, whereas 47 is inactive.
Furthermore some of the compounds showed significant anti-oxidative activity. The most active
compound in the ORAC assay was 47, which exhibited an anti-oxidative effect of 6.6 ± 1.0 TE.
However, this compound showed only weak activity during the proliferation assay (IC50 = 53.8 ±
0.3 µM) and did not inhibit tube-formation.
Figure 6. Structures of two series of nature-like acylphloroglucinols.
Chapter 1 | 19
A basic structure-activity relationship of the aliphatic mono- and bicyclic acylphloroglucinol
derivatives with short acyl side chains indicates that the in vitro anti-proliferative activity of
these acylphloroglucinols in HMEC-1 increases with increasing logP. Increasing the number of
carbon atoms in the acyl group provides higher lipophilicity, which allows the compound a better
penetration across cellular membranes in vitro assay. In contrast, the activity of the derivatives
with aromatic acylside chain depends on other properties. The molecular aspects of the observed
cellular effects are currently under investigation.
Compounds 30-34 were synthesised via Friedel-Crafts acylation (Scheme 7) with 55 to 81%
yield. The alkylation of 30-34 and geranyl bromide gave products 35-39 with moderate yields
from 55 to 60%. Finally, a para-toluenesulfonic acid (pTSA) catalysed cyclisation afforded the
target compounds 40 and 41 in 53 and 65% yield, respectively.
Scheme 7. Synthesis of acylphloroglucinol derivatives 35-41. Reagents and conditions: (a) acyl chloride,
AlCl3, CS2-PhNO2, 55°C, 2 h; (b) geranyl bromide, K2CO3, acetone, reflux overnight; (c) pTSA, benzene,
reflux, 2 h.
Amberlyst 15 efficiently catalysed the condensation of 1,3,5-trihydroxybenzene 29 with isoprene
in 53% yield in the synthesis of the second series of compounds (Scheme 8). The following
Chapter 1 | 20
Friedel-Crafts acylation gave intermediates 43-46, which were subsequently demethylated using
BBr3 to give 47-51 with 48 to 78% yield.
Scheme 8. Synthesis of acylphloroglucinol derivatives 43-51. Reagents and conditions: (a) isoprene,
Amberlyst 15, THF-Hexane; (b) benzoyl chloride, AlCl3, DCM, -5°C to r.t., overnight; (c) BBr3, DCM, -
78°C to r.t., overnight.
(-)-Epigallocatechin-3-O-gallate (EGCG)
(-)-Epigallocatechin-3-O-gallate (EGCG, 52) is the most abundant catechin in green tee
(Camellia sinensis L. KUNZE, Theaceae). It is the esterification product of epigallocatechin and
gallic acid. Many studies provide evidence that EGCG modulate multiples signal transduction
pathways controlling the unwanted proliferation of cells. It inhibits the activation of HIF-1α, NF-
κB and VEGF expression, thereby suppressing tumor angiogenesis and cancer progression.[43]
Furthermore, EGCG inhibited MMP-2 and MMP-9 (in different cell type), which seem to play
Chapter 1 | 21
an important role in tumor invasion and metastasis. Also, the inhibition of uPA by EGCG has
been observed.[44]
uPA has the ability to prevent apoptosis, stimulate angiogenesis, mitogenesis,
cell migration, and to modulate cell adhesion. The presence of the 3-galloyl moiety in catechins
led to higher biological activities,[45]
but an increasing number of aromatic hydroxyl groups
results in low stability and the inability of the compound to cross cellular membranes.[46]
To
prevent oxidation and improve its bioavailability, modifications of EGCG focus on synthesising
more stable analogues (Figure 7). Anderson et al.(2005) [47]
replaced the hydrolytically labile
ester bond with a more stable amine and amide bond and evaluated their efficacy as modulators
for β-lactam resistance in S. aureus. Landis-Piwowar.and Chi-chui Wang et al (2005) [48]
protected the hydroxy groups by peracetate. These analogues behave as prodrugs and the acetyl
group is removed by cellular cytosolic esterases. Liao (2002) and Huang (2010) et al. [49]
acylated the phenol group at 3-position, introduced fatty acids of different size and extensively
explored their structure-activity relationship to 5α-reductase. Park and coworkers (2010) [50]
also
synthesised 3-O-alkyl analogues of epicatechin. They found that the introduction of alkyl groups
instead of acyl groups enhanced antimicrobial activities and stability at pH 7.4.
Chapter 1 | 22
Figure 7. Analogues of (-)-EGCG to prevent oxidation and improve bioavailability of the compounds.
Xanthohumol
Xanthohumol (XN, 58), a naturally occurring prenylated chalcone in hop plants (Humulus
lupulus L., Cannabaceae), has been suggested to have potential to halt the development and
progression of cancer and is therefore also a compound with a chemopreventive potential.[51]
For
example, XN shows proliferative inhibition of human breast (MCF-7), colon (HT-29), and
ovarian cancer (A-2780) cells in vitro with IC50 ranging from 0.52 to 13.3 μM. Because most
cancer chemopreventive agents have also anti-angiogenic properties in vitro and in vivo, further
investigations [52]
showed that XN repressed both the NF-κB and Akt pathways in endothelial
cells, inhibited VEGF-A expression in a wound-healing assay and exhibited interference in the
angiogenic process. The first total synthesis of xanthohumol was accomplished by Erhardt et al.
in six steps with an overall yield of 10% in 2007 (Scheme 9).[53]
The method was improved by
Vogel and Heilmann et al. (2008) [54]
to yield also several xanthohumol derivatives occurring as
Chapter 1 | 23
minor compounds in hop cones or as in vivo metabolites after xanthohumol intake.[54a]
Up to now
xanthohumol in vivo metabolites are not investigated regarding their anti-agiogenic activity.
Scheme 9. Scheme 9. Synthesis of xanthohumol 58. Reagents and conditions: (a) MOMCl, diisopropyl
ethyl amine, CH2Cl2; (b) 3-methyl-2-butene-1-ol, diethylazodicarboxylate, PPh3, toluene/THF; (c) N,N-
dimethylaniline, reflux; (d) (CH3O)SO2, K2CO3, acetone, reflux; (e) 4-methoxymethylbenzaldehyde
aqueous NaOH, MeOH, reflux; (f) concentrated HCl (pH=1), MeOH/H2O, rt.
Chapter 1 | 24
Genistein
Genistein (60) is an isoflavone extracted from soybeans (Glycine max (L.) MERR, Fabaceae). It
is present as the 7-O-glycoside genistin in the plant, but during the processing of soya products a
significant amount of the aglycone genistein is liberated. Genistein was originally described as
an exclusive inhibitor of tyrosine-specific protein kinases.[55]
These kinases are responsible for
the tyrosine-specific protein phosphorylation, which is required for the regulation of cell
functions, including cell proliferation and cell transformation. Later on genistein was also found
to act as oestrogen receptor agonist.[56]
The anti-angiogenic potential of genistein was first
reported by Fotsis et al in 1993.[57]
Then further studies showed that genistein inhibited
angiogenic processes in various in vitro and in vivo models.[58] The typical synthesis of genistein
starts from m-trihydroxybenzene 29 (Scheme 10).[59]
After Houben–Hoesch reaction or Friedel-
crafts acylation, cyclization of the resulting hydroxyketone (59) in the presence of BF3-Et2O
gave genistein (60) in good yield.
Scheme 10. Synthesis of genistein 60. Reagents and conditions: (a) 4-hydroxyphenyl acetonitrile,
anhydrous HCl, ZnCl2-Et2O, then aq HCl, heat or 4-hydroxyphenylacetic acid, BF3-Et2O, 120°C; (b) BF3-
Et2O, DMF, MeSO2Cl, 100°C, 2 h.
In the Friedel-crafts acylation, BF3-Et2O was used as the catalyst and solvent. The following
formation of the pyrone was also catalysed by BF3-Et2O, a convenient one-pot synthesis of 60
was achieved without isolation of 59.[60]
Fisetin and Quercetin
The flavonoids fisetin (67) and quercetin (68) belong to the flavonol subgroup exhibiting a
double bond between C-2/C-3 and a hydroxyl group at C-3. Flavonols are the most abundant
Chapter 1 | 25
flavonoid subtype in plants which mainly occur as glycosides. Nevertheless, pharmacological
testing concentrated (historically based) on the investigation of the aglycones. This has been
often criticised, but it is most likely an important aspect with regard to flavonoid metabolism. It
has been shown that the flavonoid glycosides are not absorbed after oral intake but are cleaved
by lactase-phlorizin hydrolase and absorbed as the corresponding aglycone. The aglycone passes
the cell membrane by passive diffusion and undergoes phase-II metabolism in enterocytes and
the liver leading to glucuronides as the main metabolites. At least, a release of the aglycones
from the glucuronides in tissues or cells with -glucuronidase activity is possible.[61]
Fisetin can be found in many fruits like strawberries and apples (Malus spec. MILL., Rosaceae)
as well as in vegetables like onions (Allium cepa L., Amaryllidaceae, subfamily Allioideae
former family Alliaceae). It possesses anticancer activities in various cancer models, for example,
it can inhibit androgen receptor signalling and tumor growth in athymic nude mice,[62]
it can
cause apoptosis and cell cycle arrest in human prostate cancer LNCaP cells,[63]
in HCT-116
human colon cancer cells and it can induce apoptosis associated with an increased level of p53.[64]
P.Singh and A.Bhat et al.(2012) [65]
tested its anti-angiogenic activity for the first time. Their
study revealed that fisetin (10–50 µM) strongly inhibited the growth, proliferation and cell cycle
progression in HUVEC by down-regulating the expression of VEGF and eNOS in endothelial
cells. Another recent study also demonstrated that fisetin inhibits MMPs and reduces tumor cell
invasiveness and endothelial cell tube formation.[66]
Quercetin is besides kaempferol the most abundant aglycone in flavonol glycosides. Quercetin
glycosides occur in higher concentrations in onions, red wines, and green tea or in various
medicinal plants.[67]
In a number of early studies, quercetin showed a strong ability to inhibit
tumor growth in vivo.[68]
Quercetin, inhibits angiogenesis through multiple mechanisms such as
inhibition of COX-2 and lipoxygenase (LOX)-5, interference with the EGF receptor, the HER-2
intracellular signaling pathway, and the NF-κB nuclear transcription protein. Chen et al.(2008)
[69] reported that quercetin inhibited the proliferation of choroids-retina endothelial cells and the
migration and tube formation of RA/6A cells were also significantly inhibited by quercetin in a
dose-dependent manner, but in some cell types quercetin is also able to activate the angiogenic
pathway by inhibiting HIF-prolyl hydroxylase.[70]
Zhao et al.(2014) [71]
investigated that the anti-
angiogenic activity of quercetin in zebrafish embryos and in human umbilical vein endothelial
Chapter 1 | 26
cells (HUVECs). The formation of intersegmental vessels was disrupted in transgenic zebrafish
embryos. In HUVECs, quercetin inhibited cell viability, the expression of VEGF-2 and tube
formation dose-dependently.
The synthesis of fisetin and quercetin can be achieved by two different methods. The first choice
for synthesising fisetin and quecetin is by the Allan-Robinson reaction.[72]
However, this reaction
has some drawbacks as very harsh experimental conditions and the necessity of selective
protection and deprotection of the free hydroxyl groups with benzyl and/or benzoyl groups. An
alternative method is the Algar-Flynn-Oyamada (AFO) reaction which gives flavone-3-ols
directly, but the yield varies depending on different substrates.[73]
Simpson and co-workers (1955)
[74] improved the reaction conditions of AFO in order to synthesise the flavonol rhamnocitrin by
using bismuth carbonate and acetic acid, which increased the yields to 71% and the overall yields
to 52% over two steps. According to this, a synthesis of fisetin and quercetin with methyl
protected chalcones as starting material was proposed (Scheme 11).
Scheme 11. Synthesis of fisetin 67 and quercetin 68. Reagents and conditions: (a) 3,4-
dimethoxybenzaldehyde, KOH, DMF, 0°C; (b) BiCO3, AcOH, 2-ethoxyethanol, Δ; (c) BBr3, DCM, -78°C
→r.t.
Chapter 1 | 27
(2S)-7,2’,4’-Trihydroxy-5-methoxy-8-dimethylallyl flavanone
(2S)-7,2’,4’-Triihydroxy-5-methoxy-8-dimethylallyl flavanone (69, Figure 8) is a prenylated
flavanone isolated from Sophora flavescens by Wang and Yuan et al in 2013.[75]
It displays
inhibitory effects on cell proliferation, cell migration, cell adhesion and tube formation with the
human umbilical vein endothelial cell line ECV304, which are the four important steps in
angiogenesis process. The mechanistic study showed that compound 69 is able to regulate ROS
levels and VEGF expression in a dose-depended manner down, and induce cell cycle arrested in
G0/G1 phase.
Figure 8. Structure of (2S)-7,2’,4’-triihydroxy-5-methoxy-8-dimethylallyl flavanone 69.
Conclusions
As pathological angiogenic processes are supposed to contribute to several diseases, compounds
with anti-angiogenic activity have been intensively investigated. Besides antibodies also several
low-molecular weight compounds have been chemically and pharmacologically characterized
among them several secondary metabolites of natural origin. In anti-angiogenic strategies natural
products with phenolic substructures or belonging to the polyphenols are of special importance
as they occur in several food and medicinal plants important for human diet and health. Despite
the fact that phenolic compounds often show specific interactions in biological systems most of
them are pleiotropic substances with an effect on different cellular networks or targets. Several
natural phenolic angiogenic inhibitors like curcumin (3), epigallocatechin-3-O-gallate (52) and
xanthohumol (58) also showed remarkable chemopreventive activity. However, the stability,
availability from natural sources and bioactivity of the natural compounds are typically limited
with so far no example of very strong anti-angiogenic activity in the nano-molar range. Thus,
Chapter 1 | 28
synthetic approaches for the production, diversification and optimization are mandatory. The
modification of the structures of polyphenols improving stability and bioactivity, and further
enhancing their anti-angiogenic activity, is the main goal of current research in the field.
Analogues of the natural phenols with improved drug properties may be promising candidates
for future oncology treatment.
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Chapter 2 | 33
This chapter has been published:
Q. Sun, S. Schmidt, M. Tremmel, J. Heilmann, B. König. Eur J Med Chem. 2014, 85, 621-628.
Author contributions:
Q. Sun synthesized all the compounds and wrote the manuscript.
Chapter 2
Synthesis of natural and natural-like acylphloroglucinols with anti-
proliferative, anti-oxidative and tube-formation inhibitory activity
Abstract
Two series of natural and natural-like mono- and bicyclic acylphloroglucinols derived from
secondary metabolites in the genus Hypericum (Hypericaceae) were synthesised and tested in
vitro for anti-proliferative and tube-formation inhibitory activity in human microvascular
endothelial cells (HMEC-1). In addition, their anti-oxidative activity was determined via an
ORAC-assay. The first series of compounds (4a-e) consisted of geranylated monocyclic
acylphloroglucinols with varying aliphatic acyl substitution patterns, which were subsequently
cyclised to the corresponding 2-methyl-2-prenylchromane derivatives (5a and 5d). The second
series involved compounds containing a 2,2-dimethylchromane skeleton with differing aromatic
acyl substitution (6a-d and 7a-e). Compound 7a, (5,7-dihydroxy-2,2-dimethylchroman-6-yl)-
(3,4-dihydroxyphenyl)methanone), showed the highest in vitro anti-proliferative activity with an
Chapter 2 | 34
IC50 of 0.88 ± 0.08 µM and a remarkable anti-oxidative activity of 2.8 ± 0.1 TE from the ORAC
test. Interestingly, the high anti-proliferative activity of these acylphloroglucinols was not
associated with tube-formation inhibition. Compounds (E)-1-(3-(3,7-dimethylocta-2,6-dien-1-
yl)-2,4,6-trihydroxyphenyl)-2-methylbutan-1-one (4d) and (5,7-dihydroxy-2,2-dimethyl-
chroman-6-yl)(3,4-dimethoxyphenyl)methanone (6a) exhibited moderate to weak anti-
proliferative effects (IC50 11.0 ± 1 µM and 48.0 ± 4.3 µM, respectively) and inhibited the
capillary-like tube formation of HMEC-1 in vitro, whereas 7a was inactive. The most active
compound in the ORAC assay was 7c, which exhibited an anti-oxidative effect of 6.6 ± 1.0 TE.
However, this compound showed only weak activity during the proliferation assay (IC50 53.8 ±
0.3) and did not inhibit tube-formation.
Keywords
acylphloroglucinol; chromane; HMEC-1; anti-proliferative activity; tube formation; ORAC
Chapter 2 | 35
Introduction
Acylphloroglucinols are typical secondary metabolites biosynthetically derived from the
polyketide pathway that accumulate in Hypericaceae [1]
and Clusiaceae. Their biosynthesis
begins with three malonyl-CoA (coenzyme A) molecules reacting with an activated acyl-CoA to
form an intermediate polyketide. A Claisen-like reaction enzymatically cyclises this intermediate
to an acylphloroglucinol.[2]
Depending on the starting material, the acyl moiety of a natural
acylphloroglucinol can be aliphatic, as observed in the Genus Hypericum, or aromatic, as in the
Genus Garcinia. The structural diversity of acylphloroglucinols results mainly from substitutions
on the phloroglucinol core with several prenyl or geranyl moieties. Both substituents can cyclise
and oxidise, which results in bicyclic, tricyclic or even more complex compounds.[3]
Acylphloroglucinols have attracted attention due to their broad pharmacological activity. Some
of these compounds have been reported to exhibit anti-bacterial,[4]
cytotoxic,[5]
anti-oxidative [6]
and anti-depressant [7]
effects. Hyperforin, likely the most prominent acylphloroglucinol
derivative, has been recently reported to exhibit strong anti-proliferative effects [8]
and inhibit
angiogenesis in vivo. Several key steps of this anti-angiogenic effect have been determined
pharmacologically in vitro.[9]
Due to its instability in aqueous solutions,[10]
complex structure and
limited availability, hyperforin is neither a drug candidate nor a good model compound. Thus,
synthesising pharmacologically active acylphloroglucinol derivatives with higher stability and
better solubility is an interesting challenge.
Recently, monocyclic and bicyclic acylphloroglucinol derivatives with anti-proliferative activity
for a human microvascular endothelial cell line (HMEC-1) were isolated from H. empetrifolium
(Figure 1),[11]
[12]
a plant commonly used in traditional Greek medicine. Structurally simpler than
hyperforin, the natural monocyclic compounds 4c, 4d and 5d exhibited potent anti-proliferative
activity at low micromolar concentrations (Table 1). Due to the limited availability of the
compound in plant materials, our first step was to synthesise geranylated acylphloroglucinol, 4d,
from 3d to enable further in vitro pharmacological testing. Because non-geranylated monocyclic
acylphloroglucinols (3a-e) were inactive in the proliferation assay (data not shown), the
remarkable activity of the geranylated monocyclic compound, 4d, encouraged us to more
Chapter 2 | 36
systematically investigate this class of molecules. First, the acyl group length was varied, and the
geranyl moiety was subsequently cyclised, which provided the first series of geranylated
monocyclic and bicyclic aliphatic acylphloroglucinols 4a-e, 5a and 5d (Scheme 1). Of the 2-
methyl-2-prenyl-8yl chromane derivatives obtained, compound 5d had been previously isolated
from H. empetrifolium [12]
and H. amblycalyx,[12-13]
and 5a is a natural-like analogue with a
shortened acyl side chain.
Figure 1. Hypericum empetrifolium WILLD. (Photos by Eirini Aplada, Biologist.-M.Sc.)
A second series of compound syntheses (Scheme 2) was inspired by the aromatic acyl moieties
in several natural acylphloroglucinols with chromane skeletons.[14]
The bicyclic
acylphloroglucinols obtained (6a-d, 7a-e) bear aromatic acyl groups at C-6 in the chromane
skeleton with different hydroxyl or methoxy substitution patterns. Several pharmacologically
active natural acylphloroglucinols containing aromatic substitutions, such as rottlerin and its
derivatives [15]
or (-)-3-deoxy-MS-II,[16]
have a 2,2-dimethyl-chromane core. Therefore, we used
this skeleton to synthesise 6a-d and 7a-e instead of 2-methyl-2-prenyl-chromane, which
simplifies the structure.
Results and discussion
Synthesis
Compounds 3a-e were synthesised via a Friedel-Crafts acylation (Scheme 1) with a 55 to 81%
yield. Treating 3a-e and geranyl bromide with anhydrous potassium carbonate in acetone yielded
Chapter 2 | 37
the alkylation products 4a-e with moderate yields from 55 to 60%. Purifying the mixed
compounds was difficult because geranyl bromide reacts non-selective with the hydroxide
groups or aromatic ring to yield over three side products. Finally, catalysing the cyclisation using
para-toluenesulfonic acid (pTSA) afforded the target compounds 5a and 5d in 53 and 65% yield,
respectively. The 2D NMR analysis of the products and a comparison of their spectroscopic data
to literature values revealed that they selectively cyclised at the 2-OH position not the 4-OH
position, which was unambiguously determined by comparing the 1D NMR data to literature.
Scheme 1. Synthesis of compounds 4a-e and 5a, 5d; Reagents and conditions: (a) acyl chloride, AlCl3,
CS2-PhNO2, 55°C, 2 h; (b) geranyl bromide, K2CO3, acetone, reflux overnight; (c) pTSA, benzene, reflux, 2
h.
To synthesise the second series, Amberlyst 15 was used to efficiently catalyse the condensation
of 1,3,5-trihydroxybenzene (1) with isoprene (2) in 53% yield. The subsequent Friedel-Crafts
acylation yielded intermediates 6a-d, which were subsequently demethylated with BBr3 to form
7a-e with 48 to 78% yield. Compound 5e was prepared as an aliphatic 2,2-dimethyl-chroman-6yl
analogue of the 2-methyl-2-prenyl-chroman-8yl derivative 5d.
Chapter 2 | 38
Scheme 2. Synthesis of compounds 5e, 6a-d and 7a-e; Reagents and conditions: (a) isoprene, Amberlyst
15, THF/hexane; (b) benzoyl chloride, AlCl3, CH2Cl2, -5°C to r.t., overnight; (c) BBr3, CH2Cl2, -78°C to
r.t., overnight; (d) AlCl3, CH2Cl2, r.t.
Biological evaluation
The anti-proliferative activity of monocyclic aliphatic acylphloroglucinols (4a-e) in HMEC-1
increased with increasing logP (Table 1). The IC50 values decreased from 22.5 ± 8.7 µM (4a) to
8.7 ± 3.2 µM (4e) upon extending the acyl-side chain. The anti-proliferative activity of the
bicyclic aliphatic acylphloroglucinols (5a and 5d) had the same range, and their activity likewise
increased with the logP to yield IC50 values of 20.3 ± 5.2 and 13.3 ± 3.8 µM, respectively.
Increasing the number of carbon atoms in the acyl group provides higher lipophilicity, which
allows the compound to better penetrate and cross cellular membranes. Accordingly, changing
the 2-prenyl (in 5d) to a methyl group (in 5e) decreased the lipophilicity (5d logP 5.5 versus 5e
logP 3.77) and anti-proliferative effect (5d 13.3 ± 3.8 and 5e 29.3 ± 5.2 µM). The reduced anti-
proliferative effect for 5e matches the slight activity reduction for aliphatic chromanes acylated
Chapter 2 | 39
at C-6 relative to the C-8 acyl derivatives, as reported for natural acylphloroglucinols by Schmidt
et al.[12]
Except for compounds 7a and 7e, which exhibited a strong anti-proliferative activity with IC50
values of 0.88 ± 0.08 and 7.6 ± 1.5 µM, respectively, acylphloroglucinols in the second series
with an aromatic acyl side chain and 2,2-dimethyl-chromane-6yl core exhibited lower anti-
proliferative activities relative to their aliphatic 8yl analogues (Table 1) and the 6yl derivative
5e. The strong activity for 7a resulted from its 3,4-dihydroxy-phenyl substitution, whereas
protecting the hydroxyl groups with methyl groups or changing the hydroxyl substitution pattern
dramatically decreased the activity. Chromane 7a is 50-fold more active than the 3,4-
dimethoxy-benzoyl 6a (48.0 ± 4.3 µM) and 30-fold more active than the 3,5-dihydroxybenzoyl
derivative 7b (25.5 ± 3.4 µM). Furthermore, dimethoxylation significantly reduced the water
solubility and hampered using aqueous solutions in vitro. Thus, compound 6b, which bears a 3,
5-dimethoxy-benzoyl group was not tested further in our assays. In contrast to the aliphatic
series, the logPs for compounds 6a-d (without 6b) and 7a-e did not correlate to their anti-
proliferative activity in vitro, which implies they may act via a different mechanism than
acylphloroglucinols 4a-e, especially for the highly active compounds 7a and 7e.
The anti-oxidative activity of the acylphloroglucinols was tested via an ORAC (Oxygen Radical
Absorbance Capacity) assay. Phloroglucinols containing aromatic acyl groups generally showed
higher anti-oxidative effects (1.1 ± 0.1 – 6.6 ± 1.0 TE) relative to their monocyclic and bicyclic
counterparts with aliphatic acyl substitution (0.3 ± 0.1 – 1.2 ± 0.2 TE). We also determined the
ORAC activity of natural 8-hydroxy-6-yl chromanes, 1-[5,7-dihydroxy-2-methyl-2-(4-
methylpent-3-enyl)-chroman-6-yl]-2-methylpropan-1-one and 1-[5,7-dihydroxy-2-methyl-2-(4-
methylpent-3-enyl)-chroman-6-yl]-2-methylbutan-1-one, which were recently isolated from H.
empetrifolium [11]
, relative to the aliphatic 6-hydroxy-8-yl 5a. Their activities were 0.3 ± 0.1 and
0.4 ± 0.1 TE, which indicated that the anti-oxidative activity of the aliphatic 8-hydroxy-6-yl
chromanes was reduced further relative to the 6-hydroxy-8-yl chromanes in our series. The
strongest activity was observed for compound 7c with 6.6 ± 1.0 TE, which is comparable to
strong anti-oxidants such as caffeic acid or protocateichuic acid as reported by Davalos et al.[17]
Chapter 2 | 40
Table 1. Anti-proliferative activity of aliphatic monocyclic (4a-e), bicyclic (5a, 5d and 5e)
acylphloroglucinols and aromatic bicyclic (6a-7e) acylphloroglucinols on HMEC-1 (mean value ± SD, n =
3), status, calculated values for logP and redox-potential (Eh). Trolox equivalents (TE) were determined in
an ORAC assay (1-5 µM) for all acylphloroglucinols with the exception of 6d.
type Compound IC50 [µM] LogPa Trolox equivalents (TE)
Redox potential
[V]b
ali
ph
ati
c si
dec
hai
n
4a 22.5 ± 8.7 4.62 1.0 ± 0.1 1.06
4b 22.4 ± 3.7 5.13 0.6 ± 0.1 0.86
4c 21.0 ± 0.4 5.49 0.8 ± 0.1 0.86
4d 11.0 ± 1.2 6.00 0.4 ± 0.1 0.86
4e 8.7 ± 3.2 6.50 0.3 ± 0.1 0.86
5a 20.3 ± 5.2 4.22 0.8 ± 0.1 1.23
5d 13.3 ± 3.8 5.59 0.3 ± 0.1 1.23
5e 29.3 ± 5.2 3.77 1.2 ± 0.2 0.92
aro
mati
c si
dec
hai
n
6a 48.0 ± 4.3 4.94 1.3 ± 0.1 0.98
6c 43.4 ± 15.3 5.54 1.1 ± 0.1 0.96
6d 51.5 ± 3.2 4.42 0.95
7a 0.88 ± 0.08 3.68 2.8 ± 0.1 0.80
7b 25.5 ± 3.4 5.30 3.0 ± 0.1 0.87
7c 53.8 ± 0.3 5.07 6.6 ± 1.0 1.10
7d 23.9 ± 2.4 3.66 3.0 ± 0.4 0.75
7e 7.6 ± 1.5 4.53 2.1 ± 0.2 0.67
xanthohumol 11.4 ± 1.1 2.3 ± 0.230
Trolox 0.49
a The partition coefficient (LogP) of compounds were calculated with software ACD (Advanced Chemistry
Development)/Lab 12.0.
b determined via cyclic voltammetry.
A high redox potential and radical scavenger activity were correlated for several phenolic
compounds,[18]
we also determined the redox potential for the acylphloroglucinols using cyclic
voltammetry measurements with Trolox as the reference compound. However, there was no
correlation between anti-oxidative activity measured in the ORAC assay and redox potential
Chapter 2 | 41
within the tested acylphloroglucinols. The aliphatic 6-hydroxy-8-yl chromanes 5a and 5d
(Eh=1.23 V each) were unable to reach the lower redox potential of the Trolox reference
compound (Eh=0.49 V), and surprisingly, the 6-acyl chromanes with aromatic substitutions
typically exhibited lower redox-potentials than the aliphatic 8-yl derivatives.
1 2
3 4 5
Figure 2: Tube-formation assay using compound 4d in different concentrations: 1. negative control (cells
untreated), 2. 50 µM, 3. 25 µM, 4. 12.5 µM, 5. 6.25 µM
Interestingly, the high anti-proliferative activity of acylphloroglucinols was not associated with
tube-formation inhibition. Compound 7a, which showed the strongest activity during the
proliferative assay, was inactive during the tube-formation assay. Only compounds 4d and 6a
maintained their activity across the same concentration range in the proliferative assay in both
series. Thus, the less specific anti-proliferative effect, which is potentially connected to a
nonspecific cytotoxic activity, can be decoupled from the more specific tube-formation
inhibition. Tube-formation is inhibited by compound 4d at 50, 25, 12.5 and 6.25 µM (Figure 2)
and by compound 6a at 100, 50, 25 and 12.5 µM (Figure 3). Untreated wells and inactive
acylphloroglucinols exhibited branching and interconnectivity, but endothelial cells treated with
Chapter 2 | 42
4d and 6a were dispersed and formed small cell clumps without significant network formation.
The effect of 4d was superior to that of 6a at the same concentration.
A B
C D E
Figure 3: Tube-formation assay with of compound 6a in different concentrations: A. negative control (cells
untreated), B. 100 µM, C. 50 µM, D. 25 µM, E. 12.5 µM
Conclusions
Overall, the results suggest acylphloroglucinols containing simpler substitution patterns than
hyperforin can show strong anti-proliferative effects and remarkable tube formation inhibition,
which are not correlated. For monocyclic and bicyclic compounds containing aliphatic side-
chains, the anti-proliferative effect is influenced by the side chain length, prenyl substitution and
increasing lipophilicity. A simple correlation to logP was not observed for acylphloroglucinol
with an aromatic acyl substitution. The prerequisite for a strong activity during the ORAC assay
was the presence of an aromatic acyl-substituent especially one with a 3,4-dihydroxy-
substitution. The ORAC assay activity did not correlate to the cyclic voltammetry redox
potential.
Chapter 2 | 43
The recently described biological importance of different terpene substituents at the hydroxyl
group monocyclic acylphloroglucinols [11]
makes the systematic synthesis of a library of O-
substituted mono- and bicyclic acylphloroglucinols interesting. Furthermore, investigating the
pharmacological activity of monocyclic phloroglucinols with aromatic acyl moieties is in
progress.
Experimental
Chemistry
1H,
13C and 2D NMR spectra were obtained at 298 K using a Bruker AVANCE 300 spectrometer
(operating at 300.13 MHz for 1H and 75.47 MHz for
13C), Bruker AVANCE 400 spectrometer
(operating at 400.13 MHz for 1H and 100.62 MHz for
13C) and Bruker AVANCE 600
spectrometer (operating at 600.25 MHz for 1H and 150.93 MHz for
13C) (Bruker, Karlsruhe,
Germany). The spectra were obtained using chloroform-d (99.8%, Deutero GmbH) or methanol-
d4 (99.8%, Deutero GmbH) and referenced against non-deuterated (1H) / deuterated (
13C)
solvents. The shift values (H and C) are always given in ppm with J values in Hz. The melting
points were measured using a Stanford Research Systems OptiMelt MPA 100. The high-
resolution mass spectra were obtained using a Finnigan MAT SSQ 710 A spectrometer at 70 eV
(HREIMS, positive and negative mode) or an Agilent 6540 UHD (HRESIMS, positive and
negative mode). Automated flash chromatography was performed on a Biotage® IsoleraTM
Spektra One device. Silica gel 60 M (40-63 µm, Merck) was used for the flash column
chromatography. The starting materials and reagents were purchased from commercial suppliers
and used without further purification. The solvents were p.a. grade for the reaction mixtures and
industrial grade for the flash column chromatography. Analytical TLC was performed on silica
gel coated alumina plates (MN TLC sheets ALUGRAM® Xtra SIL G/UV254). The visualisation
was performed using UV-light (254 and 366 nm). The Log P values were calculated using
ACD/Lab 12.0 software. The redox potential experiments were performed using an
Autolab PGSTAT3202N to determine the Eh (in V).
The purity of the tested compounds was determined via analytical HPLC, Elite LaChrom (VWR,
Darmstadt) using EZChromElite 3.1.7 Software to measure the purity in percent. Column:
Chapter 2 | 44
Hibar® 2504 Purospher Star RP18e (5 μm), wavelength: peak maximum, gradient: FA 0.1%-
MeCN 95%, 5→100 (MeCN 95%) within 30 min before washing and equilibrating the column
to the starting conditions for a further 10 min, flow: 0.750 mL/min. The concentration of the
tested compounds was 100 µM, and the injection volume was 20 µl. The compound purities
were 90% or higher.
2,2-Dimethyl-5,7-dihydroxychromane (2)
The sulfonic acid cation-exchange resin Amberlyst 15 (10.0 g) was stirred in dry THF (120 mL)
while refluxing at 6575°C. Then, 1,3,5-trihydroxybenzene (5.0 g) was added to the resin and
followed with 3.0 g of isoprene in hexane (40 mL) over 2 h. The reaction mixture was stirred for
1 hour under reflux before removing the heat source and adding 100 mL of Et2O. The resin was
filtered via vacuum filtration and rinsed with acetone (75 mL). The crude product mixture was
purified via flash chromatography (PE (bp.5070°C) /EtOAc 1:1) to afford compound 2 as white
solid. Yield: 4.08 g, 53%. Mp. 156157°C (162°C[19]
); 1H NMR (300 MHz, MeOD) δ 5.86 (d, J
= 2.4 Hz, 1H), 5.73 (d, J = 2.3 Hz, 1H), 2.52 (t, J = 6.8 Hz, 2H), 1.71 (t, J = 6.8 Hz, 2H), 1.26 (s,
6H). 13
C NMR (75 MHz, MeOD) δ 157.5, 157.3, 156.5, 101.5, 96.4, 95.6, 74.9, 33.7, 27.0, 17.7.
These spectroscopic data are in accordance with the literature values.[20]
General procedure for the synthesis of 3a-e
AlCl3 (4.0 equiv) was added to a stirred phloroglucinol suspension (1.0 equiv) in CS2.
Nitrobenzene was added to the solution over 30 min. The solution was refluxed at 55°C for 30
min. Acyl chloride (1.0 equiv) dissolved in nitrobenzene was added to the reaction mixture over
30 min before heating for another 30 min. The reaction mixture was allowed to cool with stirring
and then poured into an ice-water bath. Afterwards, 3M HCl was added. The organic solvents
were removed under a reduced pressure, and the oily residue containing the acylphloroglucinol
was extracted with Et2O. After removing the Et2O, the crude product was purified via flash
chromatography (PE (bp.5070°C) / EtOAc 5:1 → 3:1).[21]
1-(2,4,6-Trihydroxyphenyl)ethanone (3a)
Chapter 2 | 45
Yield: 1.89 g, 75%. Yellow solid. Mp. 219221°C (216218°C[22]
); 1H NMR (300 MHz, MeOD)
δ 5.79 (s, 2H), 2.59 (s, 3H). The spectroscopic data are in accordance with the literature values.[23]
1-(2,4,6-Trihydroxyphenyl)propan-1-one (3b)
Yield: 826 mg, 75%. Pale yellow solid. Mp. 175176°C (174°C[24]
); 1H NMR (300 MHz, MeOD)
δ 5.79 (s, 2H), 3.06 (q, J = 7.3 Hz, 2H), 1.12 (t, J = 7.3 Hz, 3H). The spectroscopic data are in
accordance with the literature values.[23]
2-Methyl-1-(2,4,6-trihydroxyphenyl)propan-1-one (3c)
Yield: 1.35 g, 81%. Pale yellow solid. Mp. 138140°C (140°C
[24]);
1H NMR (300 MHz, MeOD)
δ 5.80 (s, 2H), 3.97 (dt, J = 13.5, 6.8 Hz, 1H), 1.12 (d, J = 6.7 Hz, 6H). The spectroscopic data
are in accordance with the literature values.[25]
2-Methyl-1-(2,4,6-trihydroxyphenyl)butan-1-one (3d)
Yield: 3.12 g, 55%. Pale yellow oil. 1H NMR (300 MHz, MeOD) δ 5.81 (s, 2H), 3.83 (sext, J =
6.7 Hz, 1H), 1.89 – 1.67 (m, 1H), 1.47 – 1.26 (m, 1H), 1.09 (d, J = 6.8 Hz, 3H), 0.87 (t, J = 7.4
Hz, 3H). The spectroscopic data are in accordance with the literature values.[21]
2-Methyl-1-(2,4,6-trihydroxyphenyl)pentan-1-one (3e)
Yield: 1.32 g, 60%. Pale yellow oil. 1H NMR (300 MHz, MeOD) δ 5.79 (s, 2H), 3.96 (dt, J =
13.2, 6.6 Hz, 1H), 1.85 – 1.63 (m, 1H), 1.43 – 1.18 (m, 3H), 1.10 (d, J = 6.7 Hz, 3H), 0.90 (t, J =
6.0 Hz, 3H). HRMS (EI-MS) calcd for C12H16O4 [M+H]+
225.1121, found 225.1123.
General procedure for synthesising 4a-e
A mixture of 3a-e (1 equiv), geranyl bromide (1 equiv), and K2CO3 (2 equiv) in acetone was
refluxed for 24 h. Evaporating the acetone, adding a 2 N HCl solution, extracting with EtOAc,
and removing the solvent were followed by a flash column chromatography on silica gel with PE
（bp.5070°C)/EtOAc (5:1) to yield the corresponding products.[26]
Chapter 2 | 46
(E)-1-(3-(3,7-Dimethylocta-2,6-dien-1-yl)-2,4,6-trihydroxyphenyl)ethanone (4a).
Yield: 385 mg, 58%. Pale yellow solid. Mp. 112114°C. 1H NMR (300 MHz, CDCl3) δ 11.41(s,
br, 1H), 8.62 (s, br, 1H), 6.35 (s, 1H), 5.86 (s, 1H), 5.25 (dd, J = 7.2, 6.0 Hz, 1H), 5.13 – 4.97 (m,
1H), 3.37 (d, J = 7.1 Hz, 2H), 2.67 (s, 3H), 2.14 – 2.04 (m, 4H), 1.81 (d, J = 0.9 Hz, 3H), 1.67 (s,
3H), 1.59 (s, 3H). 13
C NMR (75 MHz, CDCl3) δ 203.8, 162.4, 161.4, 160.4, 140.0, 132.2, 123.6,
121.4, 105.4, 150.2, 95.3, 39.7, 32.9, 26.3, 25.7, 21.5, 17.7, 16.2. HRMS (EI-MS) calcd for
C18H24O4 [M+H]+
305.1747, found 305.1745.
(E)-1-(3-(3,7-Dimethylocta-2,6-dien-1-yl)-2,4,6-trihydroxyphenyl)propan-1-one (4b).
Yield: 182 mg, 60%. Pale yellow solid. Mp. 114116°C. 1H NMR (400 MHz, CDCl3) δ 11.40 (s,
br, 1H), 8.60 (bs, 1H), 6.01 (s, 1H), 5.84 (s, 1H), 5.25 (td, J = 7.2, 1.1 Hz, 1H), 5.05 (m, 1H),
3.38 (d, J = 7.2 Hz, 2H), 3.09 (q, J = 7.2 Hz, 2H), 2.17 – 2.04 (m, 4H), 1.81 (d, J = 0.7 Hz, 3H),
1.68 (d, J = 0.6 Hz, 3H), 1.60 (s, 3H), 1.17 (t, J = 7.2 Hz, 3H). 13
C NMR (101 MHz, CDCl3) δ
206.8, 162.3, 160.7, 160.1, 140.2, 132.2, 123.6, 121.4, 105.5, 104.9, 95.4, 39.7, 37.3, 26.3, 25.7,
21.6, 17.7, 16.2, 8.6. HRMS (EI-MS) calcd for C19H26O4 [MH]- 317.1758, found 317.1752.
(E)-1-(3-(3,7-Dimethylocta-2,6-dien-1-yl)-2,4,6-trihydroxyphenyl)-2-methylpropan-1-one (4c).
Yield: 219 mg, 55%. Pale yellow solid. Mp. 109111°C. 1H NMR (400 MHz, CDCl3) δ 11.55 (s,
br, 1H), 8.35 (s, br, 1H), 5.93 (s, 1H), 5.83 (s, 1H), 5.32 – 5.20 (m, 1H), 5.12 – 4.97 (m, 1H),
3.92 – 3.84 (m, 1H), 3.38 (d, J = 6.9 Hz, 2H), 2.10 (t, J = 5.0 Hz, 4H), 1.81 (s, 3H), 1.69 (s, 3H),
1.60 (s, 3H), 1.18 (d, J = 6.7 Hz, 6H). 13
C NMR (101 MHz, CDCl3) δ 210.6, 162.6, 160.7, 159.8,
140.2, 132.2, 123.6, 121.4, 105.6, 104.2, 95.5, 39.7, 39.3, 26.3, 25.7, 21.6, 19.3, 19.3, 17.7, 16.2.
HRMS (EI-MS) calcd for C20H28O4 [MH]- 331.1915, found 331.1906.
(E)-1-(3-(3,7-Dimethylocta-2,6-dien-1-yl)-2,4,6-trihydroxyphenyl)-2-methylbutan-1-one (4d).
Yield: 214 mg, 60%. Pale yellow solid. Mp. 9697°C. 1H NMR (300 MHz, CDCl3) δ 11.67 (s,
br, 1H), 8.20 (s, br, 1H), 5.90 (s, 1H), 5.78 (s, 1H), 5.21 (t, J = 6.0 Hz, 1H), 5.11-4.91 (m, 1H),
3.75-3.64 (m, 1H), 3.33 (d, J = 7.1 Hz, 2H), 2.05 (s, br, 4H), 1.77 (s, 3H), 1.63 (s, 3H), 1.55 (s,
3H), 1.36 (tt, J = 14.5, 7.3 Hz, 2H), 1.11 (d, J = 6.8 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H). 13
C NMR
(75 MHz, CDCl3) δ 210.8, 162.7, 161.2, 160.3, 139.4, 132.1, 123.7, 121.7, 105.7, 104.7, 95.4,
Chapter 2 | 47
45.9, 39.7, 27.0, 26.3, 25.7, 21.6, 17.7, 16.7, 16.2, 12.0. HRMS (EI-MS) calcd for C21H30O4
[M+H]+ 347.2215, found 347.2217.
(E)-1-(3-(3,7-Dimethylocta-2,6-dien-1-yl)-2,4,6-trihydroxyphenyl)-2-methylpentan-1-one (4e).
Yield: 78 mg, 55%. Pale yellow solid. Mp. 7779°C. 1H NMR (300 MHz, CDCl3) δ 5.84 (s, 1H),
5.26 (t, J = 6.0 Hz, 1H), 5.14 – 4.91 (m, 1H), 3.90-3.79 (m, 1H), 3.37 (d, J = 7.1 Hz, 2H), 2.15 –
2.03 (m, 4H), 1.81 (s, 3H), 1.67 (s, 3H), 1.59 (s, 3H), 1.42 – 1.24 (m, 4H), 1.15 (d, J = 6.8 Hz,
3H), 0.89 (t, J = 6.0 Hz, 3H). 13
C NMR (75 MHz, CDCl3) δ 210.7, 162.8, 160.8, 159.6, 140.1,
132.2, 123.6, 121.5, 105.7, 104.6, 95.5, 44.2, 39.7, 36.2, 26.3, 25.7, 21.6, 20.7, 17.7, 17.2, 16.2,
14.2. HRMS (EI) calcd for C22H32O4 [MH]- 359.2228, found 359.2229.
1-(5,7-Dihydroxy-2-methyl-2-(4-methylpent-3-en-1-yl)chroman-8-yl)ethanone (5a).
Yield: 107 mg, 53%. Pale yellow oil. 1H NMR (300 MHz, CDCl3) δ 13.87 (s, 1H), 6.33 (s, 1H),
5.95 (s, 1H), 5.09 (ddd, J = 7.1, 4.1, 1.2 Hz, 1H), 2.64 (s, 3H), 2.63 – 2.55 (m, 2H), 2.10-2.02 (m,
2H), 1.95 – 1.63 (m, 5H), 1.60 (s, 3H), 1.35 (s, 3H). 13
C NMR (75 MHz, CDCl3) δ 203.6, 164.8,
160.7, 157.6, 132.2, 123.7, 106.3, 99.9, 95.2, 78.4, 39.6, 33.4, 29.4, 25.7, 24.2, 22.6, 17.6, 16.0.
HRMS (EI-MS) calcd for C18H24O4 [M+H]+ 305.1747, found 305.1749.
1-(5,7-Dihydroxy-2-methyl-2-(4-methylpent-3-en-1-yl)chroman-8-yl)-2-methylbutan-1-one (5d).
Yield: 33 mg, 65%. Pale yellow oil. 1H NMR (300 MHz, CDCl3) δ 14.12 (s, 1H), 5.97 (s, 1H),
5.09 (t, J = 6.8, 1H), 3.75 (sext, J = 6.8 Hz, 1H), 2.58 (m, 2H), 2.10 − 2.03 (m, 2H), 1.87 – 1.77
(m, 3H), 1.70 (m, 2H), 1.68 (s, 3H), 1.60 (s, 3H), 1.40 (m, 1H), 1.36 (s, 3H), 1.14 (d, J = 6.8 Hz,
3H), 0.89 (t, J = 7.4 Hz, 3H). 13
C NMR (75 MHz, CDCl3) δ 210.7, 165.0, 160.5, 157.0, 132.2,
123.6, 106.0, 100.1, 95.5, 78.4, 46.3, 39.7, 29.1, 27.0, 25.6, 24.0, 22.6, 17.5, 16.6, 16.2, 12.0.
HRMS (EI-MS) calc. for C21H30O4 [M+H]+ 347.2217, found 347.2216. The spectroscopic data
are in accordance with the literature.[11]
.
General procedure for synthesis of 5e and 6a-d.[27]
Chapter 2 | 48
Anhydrous AlCl3 (3.0 equiv) was added portionwise to a solution of 2 (1.0 equiv) in CH2Cl2.
The corresponding chloride (1.0 equiv) was then added dropwise to keep the reaction
temperature below −5°C. After stirring overnight at room temperature, the mixture was poured
into ice-water and extracted with CH2Cl2. The combined organic layer was successively washed
with saturated NaHCO3 and brine, and then dried over anhydrous Na2SO4. The corresponding
products were obtained after flash column chromatography (PE（bp.50-70°C）/EtOAc 5:1 →
3:1).
1-(5,7-Dihydroxy-2,2-dimethylchroman-6-yl)-2-methylbutan-1-one (5e).
Yield: 52 mg, 26%. Pale yellow solid. Mp. 145146°C. 1H NMR (300 MHz, CDCl3) δ 13.70 (s,
br, 1H), 6.60 (s, br, 1H), 5.74 (s, 1H), 3.81 – 3.70 (m, 1H), 2.58 (t, J = 6.8Hz, 2H), 1.78 (t, J =
6.8 Hz, 2H), 1.46 – 1.34 (m, 2H), 1.32 (s, 6H), 1.16 (d, J = 6.7 Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H).
13C NMR (75 MHz, CDCl3) δ 210.3, 164.1, 160.2, 157.7, 103.8, 101.7, 95.7, 76.0, 45.8, 32.1,
27.0, 26.7, 16.7, 16.1, 12.0. The spectroscopic data are in accordance with the literature.[28]
6-(3,4-Dimethoxy)benzyl-5,7-dihydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran (6a).
Yield: 60 mg, 68%. White solid. Mp. 127128°C. 1H NMR (600 MHz, CDCl3) δ 10.73 (s, br,
1H), 7.31 (dd, J = 8.3, 2.0 Hz, 1H), 7.23 (d, J = 1.9 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 5.90 (s,
1H), 3.94 (d, J = 14.3 Hz, 6H), 2.61 (t, J = 6.8 Hz, 2H), 1.81 (t, J = 6.8 Hz, 2H), 1.36 (s, 6H). 13
C
NMR (151 MHz, CDCl3) δ 196.0, 161.7, 160.9, 158.1, 152.9, 149.6, 131.9, 122.2, 110.9, 110.9,
103.9, 101.8, 96.9, 76.0, 56.2, 56.1, 32.1, 26.8, 16.3. HRMS (EI-MS) calcd for C20H22O6 [MH]-
357.1345, found 357.1344.
6-(3,5-Dimethoxy)benzyl-5,7-dihydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran (6b)
Yield: 200 mg, 52%. Pale yellow solid. Mp. 145147°C. 1H NMR (600 MHz, CDCl3) δ 11.02 (s,
1H), 7.28 (s, br, 1H), 6.70 (d, J = 2.3 Hz, 2H), 6.62 (t, J = 2.2 Hz, 1H), 5.87 (s, 1H), 3.83 (s, 6H),
2.60 (t, J = 6.8 Hz, 2H), 1.80 (t, J = 6.8 Hz, 2H), 1.35 (s, 6H). 13
C NMR (151 MHz, CDCl3) δ
Chapter 2 | 49
196.7, 162.4, 161.6, 158.8, 141.9, 104.5, 104.1, 103.7, 101.8, 97.1, 76.2, 55.6, 32.0, 26.7, 16.1.
HRMS (EI-MS) calcd for C20H22O6 [M+H]+ 359.1495, found 359.1491.
6-(4-Methoxy)benzyl-5,7-dihydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran (6c).
Yield: 270 mg, 48%. Pale yellow solid. Mp. 107108°C. 1H NMR (600 MHz, CDCl3) δ 10.81 (s,
1H), 7.64 (d, J = 8.8 Hz, 2H), 7.13 (s, 1H), 6.96 – 6.92 (m, 2H), 5.86 (s, 1H), 3.85 (s, 3H), 2.60 (t,
J = 6.8 Hz, 2H), 1.79 (s, 2H), 1.34 (s, 6H). 13
C NMR (151 MHz, CDCl3) δ 196.4, 163.0, 161.4,
160.8, 157.8, 132.0, 130.7, 114.1, 104.0, 101.6, 96.7, 75.9, 55.4, 32.0, 26.7, 16.2. HRMS (EI-MS)
calcd for C19H20O5 [M+H]+ 329.1386, found 329.1384.
6-(3,4,5-Trimethoxy)benzyl-5,7-dihydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran (6d).
Yield: 135 mg, 21%. White solid. Mp. 208209°C. 1H NMR (400 MHz, CDCl3) δ 11.00 (s, 1H),
7.03 (s, br, 1H), 6.88 (s, 2H), 5.89 (s, 1H), 3.91 (s, 3H), 3.88 (s, 6H), 2.62 (t, J = 6.8 Hz, 2H),
1.81 (t, J = 6.8 Hz, 2H), 1.36 (s, 6H). 13
C NMR (101 MHz, CDCl3) δ 196.3, 162.2, 161.4, 158.3,
153.9, 141.5, 134.8, 104.9, 103.7, 101.9, 97.0, 76.2, 61.0, 56.4, 32.1, 26.8, 16.2. HRMS (EI-MS)
calcd for C21H24O7 [MH]- 387.1449, found 387.1455.
General procedure for synthesis of 7a-e.[29]
BBr3 (2.5 mmol) was added to a solution of 6a-d (0.84 mmol) in DCM (30 mL) at −78°C. The
reaction mixture was stirred for 30 min, warmed to room temperature and stirred overnight under
an N2 atmosphere. The mixture was quenched by carefully pouring into iced water. The aqueous
layer was extracted three times with EtOAc and washed with 5% NaHSO3 and water before
drying the organic layer over anhydrous Na2SO4 and removing the solvent under reduced
pressure to yield the target products.
6-(3,4-Dihydroxy)benzyl-5,7-dihydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran (7a).
Chapter 2 | 50
Yield: 93 mg, 78%. Pale yellow solid. Mp: 7981°C. 1H NMR (600 MHz, MeOD) δ 7.06 (d, J =
2.0 Hz, 1H), 7.02 (dd, J = 8.2, 2.0 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 5.95 (s, 1H), 2.55 (s, 2H),
1.65 (t, J = 6.8 Hz, 2H), 1.04 (s, 6H). 13
C NMR (151 MHz, MeOD) δ 199.5, 161.6, 160.7, 156.3,
150.6, 145.5, 134.4, 123.2, 117.2, 115.3, 107.6, 101.5, 95.3, 76.0, 33.1, 26.6, 17.6. HRMS (EI-
MS) calcd for C18H18O6 [M+H]+ 331.1182, found 331.1181.
6-(3,5-Dihydroxy)benzyl-5,7-dihydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran (7b).
Yield: 38 mg, 53%. Pale yellow solid. Mp. 99101°C. 1H NMR (300 MHz, MeOD) δ 6.48 (d, J
= 2.2 Hz, 2H), 6.40 – 6.32 (m, 1H), 5.74 (s, 1H), 2.59 (t, J = 6.8 Hz, 2H), 1.80 (t, J = 6.8 Hz, 2H),
1.33 (s, 6H). 13
C NMR (75 MHz, MeOD) δ 201.1, 162.7, 162.2, 162.2, 160.1, 160.1, 159.5,
159.0, 154.2, 107.5, 106.1, 105.9, 101.6, 96.6, 76.7, 33.2, 27.0, 17.2. HRMS (EI-MS) calcd for
C18H18O6 [M+H]+ 331.1182, found 331.1174.
6-(4-Hydroxy)benzyl-5,7-dihydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran (7c).
Yield: 83 mg, 48%. Pale brown solid. Mp.157159°C. 1H NMR (300 MHz, MeOD) δ 7.60 –
7.41 (m, 2H), 6.81 – 6.63 (m, 2H), 5.77 (s, 1H), 2.59 (t, J = 6.8 Hz, 2H), 1.80 (t, J = 6.8 Hz, 2H),
1.33 (s, 6H). 13
C NMR (75 MHz, MeOD) δ 199.4, 162.5, 161.8, 161.3, 159.2, 133.7, 132.7,
115.3, 106.2, 101.8, 96.6, 76.5, 33.3, 27.0, 17.4. HRMS (EI-MS) calcd for C18H18O5 [M+H] +
315.1232, found 315.1225.
6-(3,5-Dimethoxy-4-hydroxy)benzyl-5,7-dihydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran
(7d).
Yield: 68 mg, 43%. Pale yellow solid. Mp. 182184°C. 1H NMR (300 MHz, CDCl3) δ 10.86 (s,
1H), 6.95 (s, 3H), 5.99 (s, 1H), 5.90 (s, 1H), 3.91 (s, 6H), 2.61 (t, J = 6.8 Hz, 2H), 1.80 (t, J = 6.8
Hz, 2H), 1.35 (s, 6H). 13
C NMR (75 MHz, CDCl3) δ 195.9, 161.9, 161.0, 158.0, 147.4, 138.9,
130.3, 105.3, 103.8, 101.9, 97.0, 76.1, 56.6, 32.1, 26.8, 16.2. HRMS (EI-MS) calcd for C20H22O7
[M+H]+ 375.1440, found 375.1438.
Chapter 2 | 51
6-(3,4-Dihydroxy-5-methoxy)benzyl-5,7-dihydroxy-2,2-dimethyl-3,4-dihydro-2H-1-benzopyran
(7e).
Yield: 113 mg, 64%. Pale yellow solid. Mp. 9597°C. 1H NMR (300 MHz, MeOD) δ 6.88 (dd, J
= 10.1, 1.9 Hz, 2H), 5.78 (s, 1H), 3.84 (s, 3H), 2.60 (t, J = 6.8 Hz, 2H), 1.80 (t, J = 6.8 Hz, 2H),
1.32 (d, J = 6.0 Hz, 6H). 13
C NMR (75 MHz, MeOD) δ 199.5, 161.3, 161.2, 159.0, 148.8, 145.7,
139.8, 132.8, 112.1, 106.5, 106.0, 101.8, 96.7, 76.4, 56.7, 33.3, 27.0, 17.4. HRMS (EI-MS) calcd
for C19H20O7 [M+H]+ 361.1287 found 361.1277.
Redox Potential Experiments.[30]
The potentials were recorded using a glassy carbon working electrode, platinum counter
electrode and silver wire pseudo reference electrode. All compounds were measured in CH3CN
with tetrabutyl ammonium tetrafluoroborate as the supporting electrolyte. The solvent was
degassed via vigorous argon bubbling prior to the measurements. All experiments were
performed under an argon atmosphere. Ferrocene was used as the internal reference for the
reduction and oxidation potentials.
Proliferation Assay.[31]
The proliferation assay was performed using a SV-40T transfected human microvascular
endothelial cell line (HMEC-1).[32]
The cells were incubated at 37°C under a 5% CO2 air
atmosphere with a constant humidity (95%). HMEC-1 were seeded into 96-well microplates
(100 µl, 1.5×103 cells/well) in Endothelial Cell Growth Medium (ECGM) with 10% FCS,
Supplement Mix and Antibiotics (all from Provitro). After 24 h, the medium in a reference plate
was removed, and these cells were stained with a crystal violet solution for 10 min to obtain a
baseline. The cells in the other plates were treated with increasing concentrations of each test
compound (dissolved in DMSO as a stock solution). After incubating for 72 h, the cells were
stained as previously described. After washing with distilled water, 100 µl of a soluble buffer
was added and the absorbance was measured using a Tecan Spectra Fluor Plus at 540 nm. The
IC50 values ± SD were calculated in µM using the GraphPad software (from three independent
experiments with different passages and each concentration in hexaplicates). Xanthohumol was
Chapter 2 | 52
used as the positive control. Pure ECGM containing 0.1% DMSO was used as the negative
control.
Tube Formation Assay.[31]
HMEC-1 build capillary-like structures on MatrigelTM
(BD Biosience, Heidelberg Germany),
which is a solubilised basement membrane extracted from Engelbreth-Holm-Swarm (EHS)
mouse sarcoma. After polymerising 10 µl of MatrigelTM
in µ-slides (ibidi, Martinsried Germany)
within 30 min at 5% CO2, 37°C, and 95% humidity, the gel was overlaid with 1×104 HMECs in
25 µl ECGM. The cells were either left untreated (+ 25 µl ECGM containing 0.1% DMSO) or
stimulated with 25 µl of ECGM including four concentrations of the synthesised
acylphloroglucinols in their IC50 range. After 19 h, the cells were photographed using a
PrimoVert microscope (Zeiss, Oberkochen Germany). Each experiment was performed in
triplicate. One representative picture is shown.
ORAC-fluorescein assay.
The antioxidant activity was determined via an oxygen radical absorbance capacity-fluorescein
(ORAC-FL) assay[16, 17]
as previously described.[33]
In brief, the ORAC-FL assay was performed
in a 96-well plate containing fluorescein (final concentration 300 nM) as a fluorescent probe and
using a 75 mM phosphate buffer (pH 7.4) for all dilution steps and as a reaction milieu. The
antioxidant (test compounds or Trolox, 20 µL) was incubated to different concentrations (test
compounds: 1–5 µM, Trolox: 1–8 µM) with a fluorescein solution (120 mL) at 37°C for 15 min.
The reaction began upon the addition of 60 µL AAPH (2,2’-azobis-(2-methylpropionamide)-
dihydrochloride, final concentration: 12 mM) for a final volume of 200 µL. After adding AAPH,
the fluorescence was recorded every minute in a Tecan 96-plate reader (λex 485 nm, λem 536 nm,
37°C) for 80 min. The reaction mixtures were prepared in quadruplicate, and at least four
independent assays were performed for each sample. The samples were measured at five
different concentrations (1–5 µM). Eight calibration curves were obtained for each assay using
1–8 µM Trolox as the antioxidant. The controls were measured without an antioxidant or without
AAPH and antioxidant. The ORAC values were expressed as Trolox equivalents (mean ± SD)
Chapter 2 | 53
using the standard curve calculated for each assay. The Regression coefficient between the AUC
and antioxidant concentration was calculated for all samples (r2 >0.93). Further positive control
measurements were performed using xanthohumol.
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[33] a) S. Vogel, S. Ohmayer, G. Brunner, J. Heilmann, Bioorgan Med Chem 2008, 16, 4286-4293; b)
S. Vogel, M. Barbic, G. Jurgenliemk, J. Heilmann, Eur J Med Chem 2010, 45, 2206-2213.
Chapter 2 | 55
1H and
13C NMR spectra of selected final compounds
1H and
13C NMR spectra for (E)-1-(3-(3,7-Dimethylocta-2,6-dien-1-yl)-2,4,6-trihydroxy phenyl)-
2-methylbutan-1-one (4d) (300 MHz, CDCl3)
Chapter 2 | 56
1H and
13C NMR spectra for 6-(3,5-Dimethoxy)benzyl-5,7-dihydroxy-2,2-dimethyl-3,4-dihydro-
2H-1-benzopyran (6b) (600 MHz, CDCl3)
Chapter 2 | 57
1H and
13C NMR spectra for 6-(4-Hydroxy)benzyl-5,7-dihydroxy-2,2-dimethyl-3,4-dihydro-2H-1-
benzopyran (7c) (300 MHz, MeOD)
Chapter 4 | 58
Manuscript in preparation.
Q.Sun synthesized all the compounds and wrote the chemistry part of the manuscript.
Chapter 3
Flavonoid derivatives as selective ABCC1 modulators: synthesis and
functional characterization
Abstract
A series of chromones, bearing substituted amino groups or N-substituted carboxamide moieties
in 2-position, was synthesised and characterized in cellular assays for modulation of the ABC
transporters-ABCC1 (MDCKII-MRP1 cells), ABCB1 (Kb-V1 cells) and ABCG2 (MCF-7/Topo
cells). The most potent ABCC1 modulators identified among these flavonoid-type compounds
were comparable to reversan regarding potency, but superior in terms of selectivity concerning
ABCB1 and ABCG2 (IC50 values: reversan: ABCC1, 4.3 µM; ABCB1, 5.6 µM, ABCG2, 109
µM; 2-[4-(Benzo[c][1,2,5]oxadiazol-5-ylmethyl)piperazin-1-yl]-5,7-dimethoxy-4H-chromen-4-
one (51): ABCC1: 11.3 µM; inactive at ABCB1 and ABCG2). Compound 51 was as effective as
reversan in reverting ABCC1-mediated resistance to cytostatics in MDCKII-MRP1 cells and
proved to be stable in mouse plasma. Modulators, such as compound 51, are of potential value as
pharmacological tools for the investigation of the (patho)physiological role of ABCC1.
Keywords
ABC transporters, multidrug resistance-associated protein 1, inhibitors, synthesis, flavonoid.
Chapter 3 | 59
Introduction
The superfamily of human ATP-binding cassette (ABC) proteins comprises 49 members divided
into 7 subfamilies (ABCA − ABCG).[1]
The proteins ABCB1 (p-glycoprotein, p-gp), ABCG2
(BCRP, MXR) and ABCC1 (MRP1) are amply expressed efflux pumps in various tissues related
with absorption, metabolism and excretion as lung, gut, kidney and liver. In addition, these
transporters are important components of physiological barriers such as the blood-brain barrier
and the blood-cerebrospinal fluid barrier, mediating the transport of endogenous compounds and
playing a protective role against a wide range of structurally diverse xenobiotics.[2]
ABC
transporters are known to lower the bioavailability of orally administered drugs and to limit the
access of pharmacotherapeutics to the central nervous system (CNS). Moreover, ABCB1,
ABCG2 and ABCC1 are the most prominent efflux transporters expressed in cancer cells and
related to resistance against numerous antitumor agents such as anthracyclines, vinca alcaloids or
epipodophyllotoxins, resulting in poor outcome of chemotherapy.[1c, 2-3]
This phenomenon is
known as multidrug resistance (MDR).[3]
Since its discovery[4]
in 1992, ABCC1 has been the
subject of many investigations on its role in physiological processes and MDR.[5]
Besides non-
small-lung cancer cells,[4]
ABCC1 overexpression was detected in acute myeloblastic leukemia,
acute lymphoblastic leukemia, prostate cancer, some types of breast cancer and especially
neuroblastoma in early childhood.[6]
Based on this evidence, the coadministration of selective
ABCC1 modulators and cytostatic, which is substrate of ABCC1, might improve the outcome in
cancer therapy. Several substances were described as modulators of ABCC1 (Figure 1),
including MK-571, flavonoids, imidazothiazole derivatives, dihydropyridines,
pyrrolopyrimidines, indolopyrimidines, anellated isoxazoles such as LY465803 and LY455776,
and reversan.[6-7]
Nevertheless, most of them are non-selective inhibitors or substrates of the
ABCC1 transporter.
Chapter 3 | 60
Figure 1. Structure of several ABCC1 (MRP1) modulators.
Selective modulators are required as pharmacological tools to investigate their role of ABC
transporters in health and disease, to improve access to the brain or to overcome chemoresistance.
Previously, potent and selective ABCB1 and ABCG2 modulators were developed in our
laboratory.[8]
Here we report on the synthesis and characterization of a new class of ABCC1
modulators based on the core structure of flavonoids.
Results and discussion
Chemistry
The design of the title compounds was inspired by different studies reporting flavonoids[7a, 7b]
as
substrates or inhibitors of ABC transporters and by recent studies, which used computational
approaches such as docking and pharmacophore models to elucidate structure-activity
relationships.[7e, 7f]
Chapter 3 | 61
Our synthetic strategy for compounds 31-53 (Scheme 1) follows a published procedure.[9]
Commercially available hydroxylated acetophones (1, 2) were methylated with Me2SO4 in good
yields. Ring closure of the obtained compounds (3, 4) was accomplished by treatment with CS2
in the presence of H2SO4 as a catalyst to give the chromene-2-thiones 5 and 6. Subsequent S-
alkylation with ethyl iodide under basic conditions followed by the oxidation of the
ethylsulfanyl-4-chromones (7, 8) with m-CPBA led to the corresponding sulfoxides (9, 10).
Finally, the reaction of 9-10 with appropriate N-substituted piperazines 11-28 or primary amines
29, 30 at ambient conditions afforded 31-46, 49-53 in 2045% yield. The aromatic amine 47 was
obtained in 80 % yield by reduction of 32 using Fe/HCl/H2O according to a known protocol [10]
and chromatographic purification in 80 % yield. Subsequent amidation with acetyl chloride gave
48 as a colorless solid in 46% yield.
a
Scheme 1. Synthesis of final compounds 31-53. Reagents and conditions: (a) Me2SO4, acetone, 65°C. (b)
(1) tBuOK, CS2, PhCH3, r.t; (2) 10% H2SO4, rt. (c) EtI, K2CO3, acetone, reflux. (d) mCPBA, DCM, reflux.
(e) DIPEA, N-substituted piperazines, EtOH, 80°C. (f) iron powder, conc. HCl, EtOH, reflux. (g) acetyl
chloride, Et3N, DCM, r.t.
Chapter 3 | 62
Compounds 62-65 and the reference compounds 66 and 67[11]
were synthesised as shown in
Scheme 2. In the first step, by analogy with a described procedure,[12]
Claisen condensation was
conducted yielding intermediates 55, 56 followed by acid-catalysed cyclisation reaction, which
gave esters 14, 15. After hydrolysis of the ester, products 62-67 were prepared under standard
amide coupling conditions with HBTU.[13]
Scheme 2. Synthesis of chromone-2-carboxamides 62-67. Reagents and conditions: (a) NaOEt, diethyl
oxalate. (b) conc.HCl, EtOH, reflux. (c) K2CO3, EtOH, THF, r.t. (d)HBr, HOAc, reflux. (e) DIPEA, HBTU,
DCM, r.t.
Compound 17 and 26 were commercially available; the synthesis of other analogues has already
been reported.[14]
The N-substituted piperazine analogues 11-16, 18-25 and 28 (Scheme 3) were
obtained by nucleophilic substitution reaction with piperazine gaving 45%75% yields.
Chapter 3 | 63
Scheme 3. Synthesis of N-substituted piperazine analogues 11-16, 18-25 and 28. Reagents and conditions:
(a) piperazine, K2CO3, THF, reflux.
To avoid of acylation on both NH groups, mono-protection of piperazine was performed with
tert-butyl dicarbonate. Followed by peptide coupling and deprotecting the Boc group (Scheme 4),
compound 27 was obtained as yellow crystals in a good yield.[13]
Scheme 4. Synthesis of (4-nitrophenyl) (piperazine-1-yl)methanone 27. Reagents and conditions: (a)
Boc2O, DCM, r.t; (b) DIPEA, HBTU, DCM, r.t. (c) (1) TFA, DCM, r.t; (2) 10% NaOH.
As shown in Scheme 5, 3-morpholinopropan-1-amine 30 was synthesised via nucleophilic
substitution, followed by subsequent deprotection of phthalimide by hydrazine hydrate.[15]
Chapter 3 | 64
Scheme 3. Synthesis of 3-morpholinopropan-1-amine 30. Reagents and conditions: (a) morpholine, Et3N,
DCM, reflux; (b) hydrazine hydrate, EtOH, reflux.
Biological evaluation
Inhibition of ABCC1-, ABCB1- and ABCG2-Transporters
The synthesised compounds 31-53, 62-67 and reference substances were investigated for
modulation of ABCC1, ABCB1 and ABCG2 using a calcein-AM and Hoechst 33342 efflux
assays in the microtiter plate format. The data are summarized in Table 2 and Table 3.
Table 2. Inhibition of ABCC1, ABCB1 and ABCG2 transporters by the new flavonoid-type modulators 31-
53 and reference compounds
Compoun
d
R2
ABCC1[a]
ABCB1[b]
ABCG2[c]
n IC50
µM[d]
Imax % [d,e]
IC50
µM[c]
Imax % [c,f]
IC50
µM[d]
Imax
% [d,g]
31 1 4-NO2 7.3 ± 2 38.8 ±
3.5 inactive - inactive[h] -
32 1 4-NO2 13.2 ± 2.5 130.1 ±
7.7 inactive - 87.8 ± 6.7
56.9 ±
2.5
33 2 4-NO2 10.9 ± 1.8 118.5 ±
6.4
33.0 ±
4.6
54.8 ±
3.6 36.9 ± 6.7
31.9 ±
2.7
34 1 H >100 - inactive - inactive[h] -
Chapter 3 | 65
-Table 1 continued-
35 1 3,4-F2 38.0 ± 16 140.6 ±
20 58.5 ± 6 14.3 ± 1 66.6 ± 11.3
50.4 ±
5
36 2 H 6.8 ± 4.2 44.5 ±
7.6 inactive - >100 -
37 1 4-OMe >100 - inactive - >130 [h] -
38 0 4-NO2 12.1 ± 2.2 64.1 ±
3.5 inactive -
18.3 ±
2.2[h]
15.9 ±
0.9
39 1 3-NO2 98.7 ±
39.2 [h]
149.9 ±
21.3
32.8 ±
3.9
29.2 ±
1.9 38.2 ± 6.4
44.0 ±
3.8
40 1 4-COOMe 58.3 ±
10.3
118.5 ±
11.8 >100 - >100 -
41 1 4-CN 26.7 ± 3.5 114.9 ±
5.2
110.7 ±
3.9
48.1 ±
1.2 99.7 ± 8.6
59.1 ±
4.0
42 1 2-NO2 63.8 ±
23.1[h]
82.5 ±
12.9 >100 - 44.8 ± 5.1
93.1 ±
5.7
43 1 4-SO2Me 66.4 ±
30.7
98.5 ±
31.2 inactive - inactive -
44 1 4-CF3 19.8 ± 2.6 158.6 ±
7.3 31.3 ± 83
21.8 ±
1.5 98.1 ± 28
99.2 ±
23.5
45 1 4-SCF3 20.8 ±
11.2
136.5 ±
24.6
36.6 ±
3.2 29.4 ± 1 91 ± 14.2
202 ±
32.3
46 1 4-SO2CF3 22.7 ± 5.8 98.8 ±
8.6 >100 - 92 ± 30.4
127 ±
34.8
47 1 4-NH2 >100 - inactive - inactive[h] -
48 1 4-NHAc >100 - inactive - inactive[h] -
49 1 Me inactive - inactive - inactive[h] -
50 1 4-NO2-benzoyl 30.1 19.7 ±
1.5 inactive - inactive -
51 benzo[c][1,2,5]oxadiazol-5-
ylmethyl 11.3 ± 1.8
119.6 ±
6.4 inactive - inactive -
52 2 3,4-dimethoxyphenyl >100 - inactive - >100 [h] -
53 3 Morpholin-4-yl 53.3 ±
17.8 14 ± 2.7 inactive - inactive
Reversan 4.3 ± 0.2 100 6.8 ±
0.3[f]
143.3 ±
3.3 n.d. -
Fumitremorgi
n C inactive - n.d. -
0.73 ±
0.09[i] 100
Tariquidar inactive - 0.22 ±
0.008[j]
100
0.52±
0.085[j]
69± 5
Chapter 3 | 66
[a] Calcein-AM microplate assay using ABCC1-overexpressing MDCK-MRP1 or [b] ABCB1-
overexpressing Kb-V1 cells. [c] Hoechst 33342 assay using ABCG2-overexpressing MCF-7/Topo cells. [d]
Mean values ± SEM from two to four independent experiments performed in triplicate or sextuplicate. [e]
Inhibitory effect (Imax) relative to the maximum response to reversan at a concentration of 30 µM (100%).
[f] Imax expressed as percent inhibition relative to tariquidar at a concentration of 1 µM (100%). [g] Imax
relative to FTC at a concentration of 10 µM (100%). [h] N=1. [i] Ref.[16]
[j] Ref.[17]
IC50 values were
calculated using SigmaPlot 11.0, four parameter logistic curve fitting; inactive: no transporter inhibition up
to a concentration of 100 µM; n.d. not determined.
The most potent ABCC1 inhibitors identified among the compounds 31-53 and 62-67 were
comparable with reversan in terms of IC50 values (31, 32, 36, 38, 51, 63-65). Interestingly, these
compounds were different from the reference substance regarding maximal response (Imax) and
especially, selectivity. Based on recent studies [7e, 7f]
we initially used 33 and 53 as scaffolds to
explore a new class of potential ABCC1 modulators. Chromone derivative 33 exhibited
modulator activity in the two-digit micromolar range and showed a slight preference for ABCC1
compared to ABCB1 and ABCG2. Solubility and ABCC1 selectivity were improved with
compound 32, the smaller homologue of 33, which was inactive at ABCB1 and much less potent
at ABCG2. Insertion of a carbonyl group between the phenylchromone moiety and N-substituted
piperazines led to a slight decrease in the inhibitory effect (62, 63), whereas the replacement of
the methylene linker in 32 by a carbonyl group (50) dramatically decreased the maximal
response from 130% to approximately 20%. Shortening of the ethylene spacer (33) to methylene
(32) did not influence the activity, but increased the solubility of the compound. In addition, 64
and 65 were prepared to study the contribution of a H-bond donor in position 5 of the
phenylchromone core. Compared with the compounds bearing two methoxy groups, the IC50
value at ABCC1 decreased from 8.2 µM (63) to 1.8 µM (65); however a moderate inhibitory
activity at ABCB1 or ABCG2 was observed. Changing the position of the nitro group on the
phenyl ring from para (32) to meta reduced the ABCC1 inhibitory activity (39). The shift of the
nitro group to ortho position in the phenyl ring decreased the inhibition of ABCC1, while the
activity at ABCG2 increased (42). Keeping in mind that nitro groups can be reduced in vivo,
resulting in potentially toxic reactive metabolites,[18]
we replaced the nitro group by different
substituents.
Chapter 3 | 67
Table 3. Inhibition of ABCC1-, ABCB1-, and ABCG2-transporters by the new flavonoid-type modulators
62–65 and reference compounds 66, 67
Compound
R1 R
2 R
3
ABCC1[a]
ABCB1[b]
ABCG2[c]
n IC50
µM[d]
Imax %
[d,e]
IC50
µM[c]
Imax %
[c,f] IC50
µM[d]
Imax %[d,g]
62 2 OMe OMe 4-
NO2 18.5 ± 3.4
139.4 ±
7.6
83.9 ±
11.7 37.7 ± 3.4 >300 -
63 1
OMe OMe 4-
NO2 8.2 ± 1.6 75.0 ± 3.8 58.2 ± 3.3 33.4 ± 2.0 >100 -
64 1
OMe OH 2-
NO2 8.3±1.1 68.8 ± 4.0 20 15.9 34.9 ± 2.5 90.5 ± 2.7
65 1
OMe OH 4-
NO2 1.8 ± 0.3 70.5 ± 4.8 inactive - 12 ± 1.2 23.1 ± 0.9
66 1 H OCH2Ph H >200 - 15.8 ± 1.4 34.3 ± 1.7 16.3 ±
4.7[h] 84.1 ± 9.9
67 - - - inactive - 1.9 ± 0.9 7.5 ± 0.9 1.8 ± 0.3[i] 108 ± 4.6
[a] Calcein-AM microplate assay using ABCC1-overexpressing MDCK-MRP1 or [b] ABCB1-
overexpressing Kb-V1 cells. [c] Hoechst 33342 assay using ABCG2-overexpressing MCF-7/Topo cells. [d]
Mean values ± SEM from two to four independent experiments performed in triplicate or sextuplicate. [e]
Inhibitory effect (Imax) relative to the maximum response to reversan at a concentration of 30 µM (100%).
[f] Imax expressed as percent inhibition relative to tariquidar at a concentration of 1 µM (100%). [g] I max
relative to FTC at 10 µM (100%). [h] Ref[11]
: ABCG2 inhibition reported for 66: 9.8 ± 2.5 at a
concentration of 5 µM, determined on mitoxantrone flow cytometric assay using ABCG2-transfected
HEK293 cells; [i] Ref.[11]
: ABCG2 inhibition reported for 67: IC50=0.17 µM, determined on mitoxantrone
flow cytometric assay using ABCG2-transfected HEK293 cells. IC50 values were calculated using
SigmaPlot 11.0, four parameter logistic curve fitting; inactive: inactive up to a concentration of 100 µM;
n.d. not determined.
Chapter 3 | 68
In general, electron-withdrawing groups on the phenyl ring markedly contributed to their
inhibition of ABCC1 transporter. Compounds (32, 33, 35, 41, 44, 46, 51) with strong electron
withdrawing group in para position showed higher potencies and maximum effects. In contrast,
lack of substituents or introduction of electron-donating groups(34, 36, 37 and 47) resulted in a
strong decrease in the inhibitory activity. Examples of concentration-response curves are shown
in the Figure 2.
Concentration [ µM ]
0,1 1 10 100
AB
CC
1 I
nh
ibitio
n (
% )
-20
0
20
40
60
80
100
120
140reversan
33
32
51
63
65
Figure 2. Concentration dependent inhibition of ABCC1 transporter in MDCKII-MRP1 cells (calcein-AM
assay) by reveresan, 32, 33, 51, 63, and 65. The inhibition is expressed relative to the maximal effect in the
presence of 30 µM of reversan set to 100%.
The flavanoids 66 and 67 were described as selective inhibitors of ABCG2.[11]
As the scaffold of
66 and 67 is similar to that of 31-53 and 62-65 we synthesised and investigated these substances
for comparison. Compound 66 showed negligible inhibition and 67 was inactive at
ABCC1(Table 2). In our assays systems, compound 66 inhibited ABCB1 with an IC50 value of
15.8 µM (Imax 34%). In case of 67, the potency determined with MCF-7/Topo cells was 10-fold
lower than that reported for HEK293-ABCG2 cells, whereas the selectivity of 67 for ABCG2
Chapter 3 | 69
was confirmed (no IC50 values for inhibition of ABCB1 and ABCC1 by 66 and 67 provided in
ref.).
Taken together, the maximum inhibitory effect was strongly dependent both on the substituents
(R1, R
2) at the chromone and the presence of an electron-withdrawing substituent at the phenyl
moiety, in particular NO2 or CF3. Whereas reversan was equipotent at ABCB1 and ABCC1,
several of the new modulators revealed preference for ABCC1 with highest selectivity residing
in compound 51.
Chemosensitivity and reversal of drug resistance of ABCC1 expressing MDCKII-MRP1 cells
On the basis of the results obtained from the functional efflux assays, the effect of the most
potent and selective ABCC1 modulator (51) on the proliferation as well as the ability to
overcome drug resistance of MDCKII-MRP1 cells was investigated in a kinetic crystal violet
chemosensitivity assay, using the modulator or the reference compound reversan alone and in
combination with the cytostatic etoposide, an ABCC1 substrate. The results are shown in the
Figure .
Incubation of MDCKII-MRP1 cells with reversan alone had no effect on cell prolifereation up to
a concentration of 10 µM (Figure 3A). The combination of etoposide at a weakly toxic
concentration of 1.5 µM with reversan at a concentration of 1.0 µM yielded a complete reversal
of the ABCC1-mediated resistance, which was not further enhanced by increasing the
concentration of the modulator (Figure 3B). Furthermore, the cytostatic effect of etoposide at 1.5
µM in combination with reversan was equieffective with application of etoposide alone at a
concentration of 10 µM (data not shown). By analogy to reversan, the proliferation of MDCKII-
MRP1 cells incubated with ABCC1 modulator 51 alone was not affected (Figure 3C). The
cytostatic effect of 1.5 µM etoposide was enhanced to a maximal response by combination with
the new modulator 51, already at a concentration of 1.0 µM (Figure 3D). In the same way, the
ABCC1 overexpressing MDCKII-MRP1 cells were incubated with another ABCC1 substrate,
Chapter 3 | 70
doxorubicin, at a per se nontoxic concentration of 50 nM in combination with reversan or 51,
leading to complete reversal of ABCC1-mediated chemoresistance (Figure 4).
Time of incubation (h)
0 20 40 60 80 100 120
T/C
co
rr (
%)
0
20
40
60
80
100
120
A5
80
nm
0,5
1,5
2,5
3,5
4,5
1,0
2,0
3,0
4,0
A
Time of incubation (h)
0 20 40 60 80 100 120
T/C
co
rr (
%)
0
20
40
60
80
100
120
A5
80
nm
0,5
1,5
2,5
3,5
4,5
1,0
2,0
3,0
4,0
B
Time of incubation (h)
0 20 40 60 80 100 120
T/C
co
rr (
%)
0
20
40
60
80
100
120
A5
80
nm
0,5
1,5
2,5
3,5
4,5
1,0
2,0
3,0
4,0
C
Time of incubation (h)
0 20 40 60 80 100 120
T/C
co
rr (
%)
0
20
40
60
80
100
120
A5
80
nm
0,5
1,5
2,5
3,5
4,5
1,0
2,0
3,0
4,0
D
Figure 3. Reversal of ABCC1 mediated drug resistance against etoposide and doxorubicin on proliferating
MDCKII-MRP1 cells. Effect of reference compound reversan alone (A) and in combination with 1.5 µM
etoposide (B) on proliferating MDCKII-MRP1 cells (long term exposure); vehicle (), 1.5 µM etoposide
() and reversan at different concentrations: 1 µM (), 2.5 µM (▼), 5 µM (), and 10 µM (). Effect of
compound 51 alone (C) and in combination with 1.5 µM etoposide (D) on proliferating MDCKII-MRP1
cells (long term exposure); vehicle (), 1.5 µM etoposide () and 51 at different concentrations: 1 µM
(), 2.5 µM (▼), 5 µM (), and 10 µM ().Effect of reversan alone and in combination with 50 nM
doxorubicin (E); vehicle (), 50 nM doxorubicin () and reversan at different concentrations: 1 µM (),
2.5 µM (▼), 5 µM (), and 10 µM (). Effect of compound 51 alone in combination with 50 nM
doxorubicin (F) on vehicle (), 50 nM doxorubicin () and 51 at different concentrations: 1 µM (), 2.5
µM (▼), 5 µM (), and 10 µM ().
Chapter 3 | 71
Time of incubation (h)
0 20 40 60 80 100 120
T/C
co
rr (
%)
0
20
40
60
80
100
120
A5
80
nm
0,5
1,5
2,5
3,5
4,5
1,0
2,0
3,0
4,0
A
Time of incubation (h)
0 20 40 60 80 100 120
T/C
co
rr (
%)
0
20
40
60
80
100
120
A5
80
nm
0,5
1,5
2,5
3,5
4,5
1,0
2,0
3,0
4,0
B
Figure 4. Effect of reference compound reversan in combination with 50 nM doxorrubicin (A) on
proliferating MDCKII-MRP1 cells (long term exposure); vehicle (), 50 nM doxorubicin () and reversan
at different concentrations: 1 µM (), 2.5 µM (▼), 5 µM (), and 10 µM (). Effect of compound 51
alone in combination with 50 nM doxorubicin (B) on proliferating MDCKII-MRP1 cells (long term
exposure); vehicle (), 50 nM doxorrubicin () and 51 at different concentrations: 1 µM (), 2.5 µM (▼),
5 µM (), and 10 µM ().
Stability in mouse plasma
As stability in plasma is a prerequisite for in vivo studies, the most potent modulator of our study
was investigated in mouse plasma. Compound 51 was incubated in plasma at a temperature of
37°C and aliquots were analyzed by HPLC over a period of 24 hours. As observed in Figure 5,
products of cleavage or degradation of compound 51 were not detected.
Chapter 3 | 72
12 14 16 18 20
100
200
300
plasma blank
24 h
5 h
120 min
60 min
30 min
15 min
1 min
Time (min)
Sig
na
l (
mV
)
Figure 5. Chromatograms of compound 51 incubated in mouse plasma over a period of 24 h. HPLC
analysis, UV detection at 220 nm.
Conclusions
Among the synthesised flavonoids several ABCC1 modulators were identified, which are
comparable to reversan in terms of potency, but superior regarding their selectivity over ABCB1
and ABCG2. The new compounds are capable of reverting chemoresistance of ABCC1
overexpressing cells and are expected to be of potential value as pharmacological tools for the
investigation of the (patho)physiological role of the ABCC1 transporter.
Experimental
Chemistry
1H,
13C and 2D NMR spectra were measured at 298 K on a Bruker AVANCE 300 spectrometer
(operating at 300.13 MHz for 1H and 75.47 MHz for
13C), a Bruker AVANCE 400 spectrometer
(operating at 400.13 MHz for 1H and 100.62 MHz for
13C) and a Bruker AVANCE 600
spectrometer (operating at 600.25 MHz for 1H and 150.93 MHz for
13C) (Bruker, Karlsruhe,
Germany). Spectra were measured in chloroform-d (99.8%, Deutero GmbH) or methanol-d4
(99.8%, Deutero GmbH) or DMSO-d6 (99.9%, Deutero GmbH) and referenced against
Chapter 3 | 73
undeuterated (1H) /deuterated (
13C) solvent. Shift values (H and C) were always given in ppm, J
values in Hz. Melting points were measured on a Stanford Research Systems OptiMelt MPA 100.
High-resolution mass spectra were measured on a Finnigan MAT SSQ 710A spectrometer at 70
eV (HREIMS, positive and negative mode) or recorded on an Agilent 6540 UHD (HRESIMS,
positive and negative mode). Automated flash chromatography was performed on a Biotage®
IsoleraTM
Spektra One device. Silica gel 60M (40-63 µm, Merck) for flash column
chromatography was used. Starting materials and reagents were purchased from commercial
suppliers and used without further purification. Solvents were used in p.a. grade for reaction
mixtures and in industrial grade for flash column chromatography. Analytical TLC was
performed on silica gel coated alumina plates (MN TLC sheets ALUGRAM® Xtra SIL
G/UV254). Visualization was conducted with UV-light (254 and 366 nm).
General synthetic procedure of N-substituted piperazines 11-16, 18-25 and 28.
Piperizine (3.0 equiv.) and K2CO3 were mixed in THF under reflux condition, then 68-82 was
added. After three more hours of refluxing, the reaction mixture was concentrated in vacuo and
partitioned between water and DCM. The organic phase was washed with brine and dried over
Na2SO4. After purification by flash chromatography (DCM : MeOH = 8:1) the products 11-16,
18-25 and 28 were obtained.
1-(4-Nitrobenzyl)piperazine (11).
Pale yellow solid. Yield: 353 mg, 69%. 1H NMR (300 MHz, CDCl3) δ 8.15 (d, J = 8.7 Hz, 2H),
7.50 (d, J = 7.6 Hz, 2H), 3.56 (s, 2H), 2.95 – 2.81 (m, 4H), 2.41 (s, 4H). 13
C NMR (75 MHz,
CDCl3) δ 147.1, 146.4, 129.5, 123.5, 62.7, 54.5, 46.0. The spectroscopic data are in accordance
with literature.[19]
1-(4-Nitrophenethyl)piperazine (12).
Yellow solid. Yield: 245 mg, 48%. 1H NMR (400 MHz, CDCl3) δ 8.15 (d, J = 8.7 Hz, 2H), 7.37
(d, J = 8.7 Hz, 2H), 2.84 – 2.99 (m, 6H), 2.66–2.57 (m, 2H), 2.50 (s, 2H), 1.61 (s, 3H). 13
C NMR
(101 MHz, CDCl3) δ 148.6, 146.3, 129.5, 123.6, 60.1, 54.4, 46.1, 33.3. HRMS (EI-MS)
Chapter 3 | 74
calculated for C12H17N3O2 [M+H]+ 237.1423, found 237.1421. The spectroscopic data are in
accordance with literature.[20]
1-Benzylpiperazine (13).
Colorless solid. Yield: 376 mg, 73%. 1H NMR (300 MHz, CD3OD) δ 7.13 – 7.44 (m, 5H), 3.54
(s, 2H), 3.30 (dt, J = 3.3, 1.6 Hz, 4H), 2.82 – 3.04 (m, 4H), 2.53 (s, 4H). 13
C NMR (75 MHz,
CD3OD) δ 138.2, 130.7, 129.4, 128.6, 64.1, 53.1, 45.7. The spectroscopic data are in accordance
with literature.[21]
1-(3,4-Difluorobenzyl)piperazine (14).
Pale yellow solid. Yield: 333 mg, 65%. 1H NMR (300 MHz, CDCl3) δ 7.22 – 6.96 (m, 3H), 3.42
(s, 2H), 2.99 – 2.77 (m, 4H), 2.40 (s, br, 4H). 13
C NMR (75 MHz, CDCl3) δ 151.9, 151.1, 148.7,
148.5, 135.4, 124.7, 117.6, 116.8, 62.4, 54.2, 46.0. The spectroscopic data are in accordance with
literature.[22]
1-Phenethylpiperazine (15).
Colorless solid. Yield: 380 mg, 74%. 1H NMR (300 MHz, CD3OD) δ 7.37 – 7.06 (m, 5H), 2.98 –
2.90 (m, 4H), 2.84-2.73 (m, 2H), 2.63-2.58 (m, 6H). The spectroscopic data are in accordance
with literature.[23]
1-(4-Methoxybenzyl)piperazine (16).
Yellow solid. Yield: 200 mg, 39%. 1H NMR (300 MHz, CDCl3) δ 7.24 – 7.19 (m, 2H), 6.87 –
6.82 (m, 2H), 3.79 (s, 3H), 3.42 (s, 2H), 2.92 – 2.83 (m, 4H), 2.40 (s, 4H). The spectroscopic
data are in accordance with literature.[24]
1-(3-Nitrobenzyl)piperazine (18).
Chapter 3 | 75
Pale yellow solid. Yield: 236 mg, 46%. 1H NMR (300 MHz, CD3OD) δ 8.24 (s, 1H), 8.14 (dd, J
= 8.2, 1.3 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 3.65 (d, J = 5.5 Hz, 2H),
2.96 – 2.82 (m, 4H), 2.49 (s, 4H). The spectroscopic data are in accordance with literature.[24]
Methyl 4-(piperazin-1-ylmethyl)benzoate (19).
Pale yellow solid. Yield: 210 mg, 41%. 1H NMR (300 MHz, CDCl3) δ 7.98 (d, J = 8.3 Hz, 2H),
7.40 (d, J = 8.2 Hz, 2H), 3.91 (s, 3H), 3.53 (s, 2H), 2.95 – 2.85 (m, 4H), 2.43 (s, 4H). ). The
spectroscopic data are in accordance with literature.[25]
4-(Piperazin-1-ylmethyl)benzonitrile (20).
Pale yellow solid. Yield: 282 mg, 55%. 1
H NMR (300 MHz, CDCl3) δ 7.59 – 7.47 (m, 1H), 7.38
(d, J = 8.3 Hz, 1H), 3.46 (s, 1H), 2.88 – 2.72 (m, 2H), 2.35 (s, 2H). The spectroscopic data are in
accordance with literature.[26]
1-(2-Nitrobenzyl)piperazine (21).
Pale yellow solid. Yield: 220 mg, 43%. 1
H NMR (300 MHz, CDCl3) δ 7.80 (dd, J = 8.0, 1.0 Hz,
1H), 7.62 – 7.57 (m, 1H), 7.52 (td, J = 7.5, 1.2 Hz, 1H), 7.43 – 7.33 (m, 1H), 3.77 (s, 2H), 2.90 –
2.78 (m, 4H), 2.46 – 2.32 (m, 4H). The spectroscopic data are in accordance with literature.[27]
1-(4-(Methylsulfonyl)benzyl)piperazine (22).
Pale yellow solid. Yield: 184 mg, 36%. 1H NMR (300 MHz, CDCl3) δ 7.89 (d, J = 8.3 Hz, 2H),
7.55 (d, J = 8.3 Hz, 2H), 3.57 (s, 2H), 3.05 (s, 3H), 2.92 (t, J = 6.0 Hz, 4H), 2.45 (s, 4H).13
C
NMR (100 MHz, CDCl3) δ 145.0, 139.3, 129.7, 127.4, 62.8, 54.0, 45.8, 44.6. The spectroscopic
data are in accordance with literature.[28]
1-(4-(Trifluoromethyl)benzyl)piperazine (23).
Chapter 3 | 76
Pale yellow solid. Yield: 261 mg, 51%. 1H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 8.1 Hz, 2H),
7.43 (d, J = 8.0 Hz, 2H), 3.52 (s, 2H), 2.89 (t, J = 4.0 Hz, 4H), 2.42 (s, 4H), 2.18 (s, 1H). 13
C
NMR (100 MHz, CDCl3) δ 142.4, 142.4, 129.8, 129.5, 129.2, 129.2, 128.9, 125.6, 125.2, 125.2,
125.1, 125.1, 122.9, 63.0, 54.2, 45.9. The spectroscopic data are in accordance with literature.[28]
1-(4-(Trifluoromethylthio)benzyl)piperazine (24).
Pale yellow oil. Yield: 438 mg, 86%. 1H NMR (300 MHz, CDCl3) δ 7.59 (d, J = 8.1 Hz, 2H),
7.38 (d, J = 8.2 Hz, 2H), 3.51 (s, 2H), 3.12 (s, 1H, NH), 2.99 – 2.76 (m, 4H), 2.39 (s, br, 4H). 13
C
NMR (75 MHz, CDCl3) δ 141.5, 136.3, 130.0, 62.8, 53.8, 45.7. HRMS (EI-MS) calculated for
C12H15F3N2S [M+H]+ 277.0981 found 277.0987.
1-(4-((Trifluoromethyl)sulfonyl)benzyl)piperazine (25).
Pale yellow solid. Yield: 173 mg, 34%. 1
H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 8.3 Hz, 2H),
7.65 (d, J = 8.3 Hz, 2H), 3.61 (s, 2H), 2.92 (d, J = 8.0 Hz, 4H), 2.45 (s, 4H), 2.30 (s, 1H). 13
C
NMR (75 MHz, CDCl3) δ 148.7, 130.9, 130.0, 62.7, 53.9, 45.7. HRMS (EI-MS) calculated for
C12H15F3N2O2S [M+H]+ 309.0879 found 309.0885.
5-(Piperazin-1-ylmethyl)benzo[c][1,2,5]oxadiazole (28).
Pale yellow solid. Yield: 236 mg, 46%. 1
H NMR (300 MHz, CDCl3) δ 7.77 (d, J = 9.3 Hz, 1H),
7.70 (s, 1H), 7.49 (dd, J = 9.3, 1.2 Hz, 1H), 3.56 (d, J = 0.8 Hz, 2H), 2.93 (t, J = 6.0 Hz, 4H),
2.49 (s, 4H). 13
C NMR (100 MHz, CDCl3) δ 149.3, 149.0, 142.9, 133.5, 116.1, 114.3, 63.1, 54.2,
45.9. The spectroscopic data are in accordance with literature.[29]
tert-Butyl piperazine-1-carboxylate (84).
To a solution of 1,4-piperazine (1.38 g, 16.0 mmol, 1.0 equiv.) in 40 mL DCM, a solution of
Boc2O (1.92 g, 8.8mmol, 0.55 equiv.) in 20 mL DCM was added dropwise at room temperature.
The reaction mixture was stirred overnight, and then evaporated. Then residue was dissolved in
40mL of water, and the precipitated product was collected by filtration. The filtrate was extracted
Chapter 3 | 77
with DCM three times and the combined organic fraction was dried over Na2SO4 and evaporated
to obtain N-Boc piperazine. Colorless solid. Yield: 1.01 g, 34%. 1H NMR (300 MHz, CDCl3) δ
3.47 – 3.32 (m, 4H), 2.86 – 2.76 (m, 4H), 1.95 (s, 1H), 1.46 (s, 9H). 13
C NMR (75 MHz, CDCl3)
δ 45.8, 28.4. The spectroscopic data are in accordance with literature.[30]
tert-Butyl 4-(4-nitrobenzyl)piperazine-1-carboxylate (85).
4-Nitrobenzoic acid (674 mg, 4.03 mmol, 1.5 equiv.), DIPEA (3.46 g, 26.8 mmol, 10.0 equiv.)
and HBTU (3.05 g, 8.04 mmol, 3.0 equiv.) were dissolved in dry DCM under N2 atmosphere and
cooled to 0°C. N-Boc piperazine (500 mg, 2.68 mmol, 1.0 equiv.) was added portion wise. Then
the reaction mixture was heated up to room temperature and stirred for 24 h. The organic phase
was washed with water and brine, dried over Na2SO4 and concentrated. The further purification
was done by column chromatography (PE (50−70°C) : EtOAc = 1: 1). Yellow solid. Yield: 321
mg, 69%.1H NMR (300 MHz, CDCl3) δ 8.45 – 8.15 (m, 2H), 7.70 – 7.48 (m, 2H), 3.51 (m, 8H),
1.47 (s, 9H). The spectroscopic data are in accordance with literature.[31]
(4-Nitrophenyl) (piperazine-1-yl)methanone (27).
To a solution of tert-butyl 4-(4-nitrobenzyl)piperazine-1-carboxylate 85 (330 mg, 0.98 mmol, 1
equiv.) in 2.5 mL DCM, 2.5 mL TFA was added dropwise at room temperature. The reaction
mixture was stirred overnight, and then evaporated to obtain the trifluoroacetic acid salt. Then 10%
NaOH was added and extracted three times with EtOAc to give (4-nitrophenyl)(piperazine-1-
yl)methanone 27. Yellow solid. Yield: 203 mg, 94%. 1H NMR (300 MHz, CDCl3) δ 8.39 – 8.22
(m, 2H), 7.70 – 7.49 (m, 2H), 3.78 (s, 2H), 3.34 (s, 2H), 2.97 (s, 2H), 2.78 (s, 2H).The
spectroscopic data are in accordance with literature.[32]
2-(3-Morpholinopropyl)isoindoline-1,3-dione (87).
In a 100 mL flask 2-(3-bromopropyl)isoindoline-1,3-dione (86, 1.0 g, 3.73 mmol, 1.0 equiv.),
morpholine (325 mg, 3.73 mmol, 1.0 equiv.) and triethylamine (906 mg, 8.96 mmol, 2.4 equiv.)
were dissolved in 25 mL of DCM. This mixture was refluxed for 30 h at 40°C and purified by
chromatography column (PE (50−70°C) : EtOAc = 1:1) to obtain product 87. Yellow oil. Yield:
1.23 g, 58%. 1H NMR (300 MHz, CDCl3) δ 7.84 (dd, J = 5.4, 3.1 Hz, 2H), 7.71 (dd, J = 5.5, 3.1
Chapter 3 | 78
Hz, 2H), 3.78 (t, J = 6.9 Hz, 2H), 3.60 – 3.44 (m, 4H), 2.47 – 2.24 (m, 6H), 1.86 (p, J = 6.8 Hz,
2H). The spectroscopic data are in accordance with literature.[32]
3-Morpholinopropan-1-amine (30).
To a round bottom flask 87 (1.0 g, 3.65 mmol, 1.0 equiv.) and hydrazine hydrate (292 mg, 9.13
mmol, 2.5 equiv.) were added and dissolved in 60 mL of ethanol. After refluxing at 80°C
overnight, the solid was filtered and the filtrate was evaporated. Finally the crude product was
dissolved in DCM and concentrated in vacuo, giving compound 30, which can be used directly
in the next step without further purification. Yield: 432 mg, 82%. 1H NMR (300 MHz, CDCl3) δ
3.75 – 3.62 (m, 4H), 2.75 (t, J = 6.8 Hz, 2H), 2.47 – 2.32 (m, 6H), 1.63 (dt, J = 14.0, 6.9 Hz, 2H).
13C NMR (75 MHz, CDCl3) δ 67.0, 56.9, 53.8, 40.7, 30.1. The spectroscopic data are in
accordance with literature.[33]
General synthetic procedure for intermediates 3 and 4.
A round bottom flask was charged with 1 or 2 (1.0 equiv.) and potassium carbonate (1.0 equiv.
or 2.0 equiv.). After addition of acetone (C1 or C2 = 0.1mol∙L-1
), the solution was refluxed at
65°C. Then dimethyl sulfate (1.0 equiv. or 2.0 equiv.) was added in three portions every 2 h. The
temperature was decreased to 40°C and the reaction mixture was stirred overnight, the solid was
filtered off and the filtrate was evaporated in vacuo. The residue was dissolved in DCM and
filtered again. After concentration, product 1-(2-hydroxy-6-methoxyphenyl)ethanone 3 or 1-(2-
hydroxy-4,6-dimethoxyphenyl)ethanone 4 were obtained as a yellow solid.
1-(2-Hydroxy-6-methoxyphenyl)ethanone (3).
Yellow solid. Yield: 1.66 g, 76%. 1H NMR (300 MHz, CDCl3) δ 13.26 (s, 1H), 7.35 (t, J = 8.4
Hz, 1H), 6.57 (dd, J = 8.4, 0.9 Hz, 1H), 6.42 – 6.35 (m, 1H), 3.90 (s, 3H), 2.68 (s, 3H). The
spectroscopic data are in accordance with literature.[34]
1-(2-Hydroxy-4,6-dimethoxyphenyl)ethanone (4).
Yellow solid. Yield: 5.72 g, 98%. 1H NMR (300 MHz, CDCl3) δ 14.03 (d, J = 0.8 Hz, 1H), 6.04
(dd, J = 3.1, 2.4 Hz, 1H), 5.90 (t, J = 2.3 Hz, 1H), 3.84 (s, 3H), 3.80 (s, 3H), 2.59 (s, 3H). 13
C
Chapter 3 | 79
NMR (75 MHz, CDCl3) δ 203.2, 167.6, 166.1, 162.9, 106.0, 93.5, 90.7, 55.5, 33.0. The
spectroscopic data are in accordance with literature.[35]
General synthetic procedure for intermediates 5 and 6.
A solution of potassium tert-butoxide (3.2 equiv.) was slowly treated with a mixture of 3 or 4
(1.0 equiv.) and CS2 (1.0 equiv.) in toluene at 10°C under nitrogen atmosphere. The solution was
stirred 16 h at room temperature. Then the mixture was extracted with water and the aqueous
phase was washed with ether. The solution was brought with 10 % sulfuric acid to pH 4 and
stirred at room temperature for 16 h under nitrogen atmosphere. Finally the solution was
extracted with DCM und the organic phase was dried over Na2SO4. After concentration, 4-
hydroxy-5-methoxy-2H-chromene-2-thione 5 or 4-hydroxy-5,7-dimethoxy-2H-chromene-2-
thione 6 were obtained as a yellow solids.
4-Hydroxy-5-methoxy-2H-chromene-2-thione (5).
Yellow solid. Yield: 714 mg, 38%. 1H NMR (300 MHz, CDCl3) δ 7.55 (t, J = 8.4 Hz, 1H), 7.17
(d, J = 8.5 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 6.75 (s, 1H), 4.10 (s, 3H). 13
C NMR (75 MHz,
CDCl3) δ 197.8, 161.1, 158.4, 156.3, 133.4, 111.4, 110.1, 106.3, 77.2, 57.3. HRMS (EI-MS)
calculated for C10H8O3S [M+H]+
209.0267, found 209.0270.
4-Hydroxy-5,7-dimethoxy-2H-chromene-2-thione (6).
Yellow solid. Yield: 2.87 g, 43%. 1H NMR (300 MHz, DMSO-d6) δ 6.74 (d, J = 2.2 Hz, 1H),
6.53 (d, J = 2.2 Hz, 1H), 6.46 (s, 1H), 3.87 (d, J = 2.0 Hz, 6H). 13
C NMR (75 MHz, DMSO-d6) δ
194.7, 164.1, 163.8, 160.5, 158.6, 106.3, 100.6, 96.4, 93.1, 56.4, 56.1. HRMS (EI-MS)
calculated for C11H10O4S [M+H]+
240.0404, found 240.0403.
General synthetic procedure for intermediates 7 and 8.
A mixture of 5 or 6 (1.0 equiv.), potassium carbonate (1.13 equiv.) and iodoethane (3.6 equiv.) in
acetone was heated to reflux for 2 h. Then the solution was concentrated in vacuo, and the
residue was partitioned between water and DCM. The aqueous phase was extracted with DCM,
dried over Na2SO4 and evaporated in vacuo to yield product 7 or 8.
2-(Ethylthio)-5-methoxy-4H-chromen-4-one (7).
Chapter 3 | 80
Brown solid. Yield: 708 mg, 96%. 1
H NMR (300 MHz, CDCl3) δ 7.51 (t, J = 8.4 Hz, 1H), 6.96
(dd, J = 8.5, 0.7 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 6.19 (s, 1H), 3.97 (s, 3H), 3.03 (q, J = 7.4 Hz,
2H), 1.42 (t, J = 7.4 Hz, 3H). 13
C NMR (75 MHz, CDCl3) δ 176.2, 166.2, 159.8, 158.3, 133.5,
110.3, 109.5, 106.8, 89.6, 56.6, 25.5, 14.2. HRMS (EI-MS) calculated for C12H12O3S [M+∙
]
236.0507, found 236.0505.
2-(Ethylthio)-5,7-dimethoxy-4H-chromen-4-one (8).
Brown solid. Yield: 2.77 g, 99%. 1H NMR (300 MHz, CDCl3) δ 6.39 (d, J = 2.3 Hz, 1H), 6.32 (d,
J = 2.3 Hz, 1H), 6.09 (s, 1H), 3.90 (d, J = 4.6 Hz, 3H), 3.85 (s, 3H), 2.99 (q, J = 7.4 Hz, 2H),
1.40 (t, J = 7.4 Hz, 3H). 13
C NMR (75 MHz, CDCl3) δ 175.4, 165.2, 163.7, 160.9, 160.4, 110.4,
108.6, 96.2, 92.5, 56.5, 55.8, 25.5, 14.2. HRMS (EI-MS) calculated for C13H14O4S [M+H]+
267.0686, found 267.0687.
General synthetic procedure for intermediates 9 and 10.
A mixture of 7 or 8 (1.0 equiv.) and m-chloroperbenzoic acid (5.0 equiv.) in DCM was refluxed
for 3 h, then cooled to −15°C and filtered. The filtrate was collected and washed with sat.
Na2SO3 and sat. NaHCO3, concentrated in vacuo to give product 9 or 10 as light yellow solid.
3-(Ethylsulfonyl)-5-methoxy-4H-chromen-4-one (9).
Yellow solid. Yield: 556 mg, 70%. 1H NMR (300 MHz, CDCl3) δ 7.66 (t, J = 8.5 Hz, 1H), 7.11
(dd, J = 8.5, 0.6 Hz, 1H), 6.97 (s, 1H), 6.90 (d, J = 8.3 Hz, 1H), 4.00 (s, 3H), 3.34 (q, J = 7.5Hz,
2H), 1.42 (t, J = 7.5Hz, 3H). 13
C NMR (75 MHz, CDCl3) δ 176.6, 159.8, 157.8, 157.5, 135.3,
115.2, 114.7, 110.1, 107.8, 56.7, 47.4, 6.8. HRMS (EI-MS) calculated for C12H12O5S [M+H]+
269.0478, found 269.0488.
2-(Ethylsulfonyl)-5,7-dimethoxy-4H-chromen-4-one (10).
Yellow solid. Yield: 2.18 g, 78%.1H NMR (300 MHz, CDCl3) δ 6.91 (s, 1H), 6.54 (d, J = 2.3 Hz,
1H), 6.42 (d, J = 2.3 Hz, 1H), 3.94 (s, 3H), 3.90(s, 3H), 3.32 (q, J = 7.5 Hz, 2H), 1.41 (t, J = 7.5
Hz, 3H). 13
C NMR (75 MHz, CDCl3) δ 175.2, 165.2, 161.2, 159.5, 156.9, 115.6, 109.5, 97.3,
93.0, 56.6, 56.0, 47.4, 6.9. HRMS (EI-MS) calculated for C13H14O6S [M+H]+
299.0584, found
299.0590.
Chapter 3 | 81
Ethyl 5,7-dimethoxy-4-oxo-4H-chromene-2-carboxylate (57).
Sodium (1.14 g, 49.4 mmol, 4.85 equiv.) was dissolved in absolute ethanol (60 mL) and 2'-
hydroxy-4', 6’-dimethoxyacetophenone 4 (2.0 g, 10.2 mmol, 1.0 equiv) and diethyl oxalate (3.6
mL, 26.5 mmol, 2.6 equiv.) was added. The mixture was stirred for 2 h under reflux conditions.
A large amount of solid appeared shortly after heating. The reaction mixture was cooled and 6N
HCl (5 mL) was added. The mixture was concentrated, diluted with water (30 mL) and extracted
with DCM. The organic layer was dried over anhydrous Na2SO4. The residue 55 was dissolved in
ethanol (60 mL) and treated with conc.HCl (6 mL) overnight. The solution was concentrated in
vacuo. Purification by chromatography (PE (50−70°C) : EtOAc = 1:1) provided compound 57.
Colorless solid. Yield: 1.56 g, 55%. 1H NMR (600 MHz, CDCl3) δ 6.95 (s, 1H), 6.59 (d, J = 2.3
Hz, 1H), 6.37 (d, J = 2.3 Hz, 1H), 4.42 (q, J = 7.2 Hz, 2H), 3.93 (s, 3H), 3.88 (d, J = 5.5 Hz, 3H),
1.40 (t, J = 7.1 Hz, 3H). 13
C NMR (151 MHz, CDCl3) δ 177.1, 164.8, 160.9, 160.6, 159.7, 149.8,
116.7, 110.1, 96.8, 93.1, 62.8, 56.4, 55.9, 14.1. The spectroscopic data are in accordance with
literature.[36]
Ethyl 5-(benzyloxy)-4-oxo-4H-chromene-2-carboxylate (58).
Sodium (1.087 g, 47.24 mmol, 4.85 equiv.) was dissolved in absolute ethanol (60 mL) and 1-(2-
(benzyloxy)-6-hydroxyphenyl)ethanone 54 (2.36 g, 9.74 mmol, 1.0 equiv.) and diethyl oxalate
(2.62 mL, 25.3 mmol, 2.6 equiv.) was added. The mixture was stirred for 2 h under reflux
condition. A large amount of solid appeared shortly after heating. The reaction mixture was
cooled and 6N HCl (4.65 mL) was added. The mixture was concentrated, diluted with water
(26.5 mL) and extracted with DCM. The organic layer was dried over anhydrous Na2SO4. The
residue 56 was dissolved in ethanol (53 mL) and treated with conc.HCl (5.3 mL) overnight. The
solution was concentrated in vacuo. Purification by chromatography (PE (50−70°C) : EtOAc =
1:1) provided compound 58. Beige solid. Yield: 2.15 g, 68%. 1H NMR (300 MHz, CDCl3) δ 7.65
– 7.50 (m, 3H), 7.46 – 7.23 (m, 4H), 7.16 (dd, J = 8.5, 0.7 Hz, 1H), 7.00 (s, 1H), 5.28 (s, 2H),
4.45 (q, J = 7.1 Hz, 2H), 1.43 (t, J = 7.1 Hz, 3H).13
C NMR (75 MHz, CDCl3) δ 177.8, 160.7,
158.6, 158.0, 136.3, 134.6, 128.7, 127.8, 126.6, 116.5, 110.9, 108.7, 70.9, 62.9, 14.1. The
spectroscopic data are in accordance with literature.[37]
Chapter 3 | 82
6,8-Dimethoxy-4-oxo-4H-chromene-2-carboxylic acid (59).
In a 500 mL flask ethyl 5,7-dimethoxy-4-oxo-4H-chromene-2-carboxylate 57 (1.5 g, 5.39 mmol,
1.0 equiv.) was dissolved in 120 mL of THF and 30 mL of ethanol. Afterwards the solution of
potassium carbonate (2.24 g, 16.17 mmol, 3.0 equiv.) in 60 mL of water was slowly added, the
mixture was stirred for 24 h at room temperature. Then 30 mL of water and 2N HCl were added
until pH≈2. After evaporating the solvents, the residue was filtered, washed with 50 mL of water
and dried at 45°C in the oven to give product 59. Colorless solid. Yield: 1.29 g, 96%. 1H NMR
(600 MHz, DMSO-d6) δ 6.69 (d, J = 2.3 Hz, 1H), 6.62 (s, 1H), 6.52 (d, J = 2.3 Hz, 1H), 3.88 (s,
3H), 3.82 (s, 3H). 13
C NMR (151 MHz, DMSO-d6) δ 175.5, 164.4, 161.4, 160.4, 159.0, 150.7,
115.3, 109.1, 96.7, 93.3, 56.14, 54.9. The spectroscopic data are in accordance with literature.[38]
5-(Benzyloxy)-4-oxo-4H-chromene-2-carboxylic acid (60).
In a 500 mL flask 58 (2.15 g, 6.63 mmol, 1.0 equiv.) was dissolved in 147 mL of THF and 38
mL of ethanol. Afterwards the solution of potassium carbonate (2.75 g, 19.89 mmol, 3.0 equiv.)
in 74 mL of water was slowly added, the mixture was stirred for 24 h at room temperature. Then
40 mL of water and 2N HCl were added until pH≈2. After evaporating the solvents, the residue
was filtered, washed with water and dried at 45°C in the oven to give product 60. Colorless solid.
Yield: 1.66 g, 85%. 1H NMR (300 MHz, DMSO-d6) δ 7.74 (t, J = 8.4 Hz, 1H), 7.62 (d, J = 7.2
Hz, 2H), 7.48 – 7.24 (m, 3H), 7.17 (dd, J = 26.2, 8.1 Hz, 2H), 6.74 (s, 1H), 5.27 (s, 2H). The
spectroscopic data are in accordance with literature.[39]
5-Hydroxy-7-methoxy-4-oxo-4H-chromene-2-carboxylic acid (61).
Compound 59 (500 mg, 2.00 mmol, 1.0 equiv.) was dissolved in 5 mL of HBr and 10 mL HOAc
(0.13mol L-1
of compound 59) and the reaction mixture was refluxed for 2 h. After cooling, the
solution was extracted with ethyl acetate (30 mL × 3), washed three times with water (30 mL × 3)
and brine (30 mL × 3) and dried over Na2SO4. After evaporation compound 61 was obtained.
Yellow solid. Yield: 237 mg, 50%. 1H NMR (600 MHz, CD3OD) δ 6.92 (s, 1H), 6.64 (d, J = 2.3
Hz, 1H), 6.39 (d, J = 2.3 Hz, 1H), 3.89 (s, 3H). The spectroscopic data are in accordance with
literature.[40]
General synthetic procedure for final compounds 31-53.
Chapter 3 | 83
To a solution of 9 or 10 (1.0 equiv.) in ethanol N-subtituted piperazine derivatives (1.0 equiv.)
and DIPEA (5.0 equiv.) were added at 0 °C. Then the mixture was refluxed for 24 h at 80°C.
After purification by flash column, the final products 31-53 were obtained in moderate yields.
5-Methoxy-2-(4-(4-nitrobenzyl)piperazin-1-yl)-4H-chromen-4-one (31).
Pale yellow solid. Yield: 119 mg. 54%. Mp. 167−168°C. 1H NMR (400 MHz, CDCl3) δ 8.24 –
8.17 (m, 2H), 7.54 (d, J = 8.7 Hz, 2H), 7.43 (t, J = 8.4 Hz, 1H), 6.87 (dd, J = 8.4, 0.8 Hz, 1H),
6.77 (d, J = 8.0 Hz, 1H), 5.44 (s, 1H), 3.96 (s, 3H), 3.66 (s, 2H), 3.55 – 3.45 (m, 4H), 2.69 – 2.50
(m, 4H). 13
C NMR (101 MHz, CDCl3) δ 177.8, 161.1, 159.6, 155.9, 147.4, 145.4, 132.2, 129.5,
123.7, 113.1, 108.9, 106.8, 88.9, 61.9, 56.5, 52.2, 44.5. HRMS (EI-MS) calculated for
C21H21N3O5 [M+H]+ 396.1554, found 396.1555.
5,7-Dimethoxy-2-(4-(4-nitrobenzyl)piperazin-1-yl)-4H-chromen-4-one (32).
White solid. Yield: 53 mg, 37%. Mp. 190−192°C.1H NMR (300 MHz, CDCl3) δ 8.19 (d, J = 8.6
Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H), 6.32 (q, J = 2.4 Hz, 2H), 5.33 (s, 1H), 3.90 (s, 3H), 3.84 (s,
3H), 3.64 (s, 2H), 3.48 – 3.41 (m, 4H), 2.61 – 2.46 (m, 4H). 13
C NMR (75 MHz, CDCl3) δ 177.6,
162.9, 161.1, 160.6, 157.4, 147.3, 145.5, 129.5, 123.7, 107.4, 95.8, 92.4, 88.6, 61.9, 56.4, 55.6,
52.2, 44.62. HRMS (EI-MS) calculated for C22H23N3O6 [M+H]+
426.1660, found 426.1663.
5,7-Dimethoxy-2-(4-(3-nitrophenethyl)piperazin-1-yl)-4H-chromen-4-one (33).
White solid. Yield: 41 mg, 28%. Mp. 197−198°C. 1H NMR (400 MHz, CDCl3) δ 8.17 (t, J = 8.3
Hz, 2H), 7.38 (d, J = 8.6 Hz, 2H), 6.35 – 6.33 (m, 2H), 5.35 (s, 1H), 3.92 (s, 3H), 3.85 (s, 3H),
3.50 – 3.37 (m, 4H), 3.00 – 2.86 (m, 2H), 2.72-2.67 (m, 2H), 2.65 – 2.58 (m, 4H). 13
C NMR (75
MHz, CDCl3) δ 177.6, 162.9, 161.1, 160.6, 157.4, 147.8, 146.6, 129.5, 123.7, 107.5, 95.8, 92.4,
88.6, 59.1, 56.5, 55.6, 52.2, 44.5, 33.3. HRMS (EI-MS) calculated for C23H25N3O6 [M+H]+
440.1816, found 440.1815.
2-(4-Benzylpiperazin-1-yl)-5,7-dimethoxy-4H-chromen-4-one (34).
Pale yellow solid. Yield: 40 mg, 31%. Mp.90−92°C. 1H NMR (300 MHz, CD2Cl2) δ 7.37 – 7.22
(m, 5H), 6.38 (d, J = 2.3 Hz, 1H), 6.32 (d, J = 2.3 Hz, 1H), 5.32 (dd, J = 2.9, 1.8 Hz, 1H), 3.86 (s,
3H), 3.84 (s, 3H), 3.54 (s, 2H), 3.47 – 3.39 (m, 4H), 2.58 – 2.48 (m, 4H). 13
C NMR (75 MHz,
CD2Cl2) δ 177.1, 163.3, 161.7, 160.9, 157.9, 138.4, 129.5, 128.7, 127.6, 107.7, 96.0, 92.8, 88.3,
Chapter 3 | 84
63.1, 56.5, 56.1, 52.5, 45.2. HRMS (EI-MS) calculated for C22H24N2O4 [M+H]+ 381.1809, found
381.1811.
2-(4-(3,4-Difluorobenzyl)piperazin-1-yl)-5,7-dimethoxy-4H-chromen-4-one (35).
White solid. Yield: 32 mg, 23%. Mp.159−161°C. 1H NMR (300 MHz, CDCl3) δ 7.23 – 6.99 (m,
3H), 6.36 – 6.27 (m, 2H), 5.34 (s, 1H), 3.92 (s, 3H), 3.85 (s, 3H), 3.50 (s, 2H), 3.47 – 3.39 (m,
4H), 2.57 – 2.47 (m, 4H). 13
C NMR (75 MHz, CDCl3) δ 177.6, 162.9, 161.1, 160.6, 157.4, 124.6,
117.4, 117.1, 107.9, 95.74, 92.4, 88.6, 61.7, 56.5, 55.6, 52.1, 44.6. HRMS (EI-MS) calculated for
C22H22F2N2O4 [M+H]+ 417.1620, found 417.1624.
5,7-Dimethoxy-2-(4-phenethylpiperazin-1-yl)-4H-chromen-4-one (36).
Yellow solid. Yield: 36 mg, 27%. Mp. 145−147°C. 1H NMR (300 MHz, CD3OD) δ 7.35 – 7.10
(m, 5H), 6.57 (d, J = 2.4Hz, 1H), 6.45 (d, J = 2.3 Hz, 1H), 5.36 (s, 1H), 3.87 (d, J = 4.2 Hz, 6H),
3.63 – 3.53 (m, 4H), 2.88-2.83 (m, 2H), 2.74 – 2.64 (m, 6H). 13
C NMR (75 MHz, CD3OD) δ
180.1, 165.4, 163.1, 161.7, 158.8, 141.0, 129.8, 129.6, 127.3, 107.2, 97.1, 93.9, 87.7, 61.2, 56.5,
53.3, 45.3, 34.0. HRMS (EI-MS) calculated for C23H26N2O4 [M+H]+
395.1965, found 395.1958.
5,7-Dimethoxy-2-(4-(4-methoxybenzyl)piperazin-1-yl)-4H-chromen-4-one (37).
White solid. Yield: 50 mg, 36%. Mp. 120−122°C. 1H NMR (300 MHz, CDCl3) δ 7.23 (d, J = 8.6
Hz, 2H), 6.90 – 6.84 (m, 2H), 6.32 (q, J = 2.3 Hz, 2H), 5.33 (s, 1H), 3.93 – 3.88 (m, 3H), 3.84 (s,
3H), 3.81 (s, 3H), 3.49 (s, 2H), 3.46 – 3.39 (m, 4H), 2.58 – 2.43 (m, 4H). 13
C NMR (75 MHz,
CDCl3) δ 177.6, 162.9, 161.2, 160.6, 158.9, 157.4, 130.4, 129.4, 113.7, 107.5, 95.7, 92.4, 88.4,
62.3, 56.4, 55.6, 55.3, 52.0, 44.6. HRMS (EI-MS) calculated for C23H26N2O5 [M+H]+
411.1914,
found 411.1918.
5,7-Dimethoxy-2-(4-(4-nitrophenyl)piperazin-1-yl)-4H-chromen-4-one (38).
White solid. Yield: 36 mg, 26%. Mp. 203−205°C. 1H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 9.4
Hz, 2H), 6.86 (d, J = 9.4 Hz, 2H), 6.38 − 6.35 (m, 2H), 5.41 (s, 1H), 3.93 (s, 3H), 3.87 (s, 3H),
3.66-3.64 (m, 4H), 3.60 − 3.57 (m, 4H). 13
C NMR (101 MHz, CDCl3) δ 177.4, 163.1, 160.8,
160.7, 157.40, 154.2, 139.3, 126.0, 113.0, 107.5, 95.9, 92.5, 88.8, 56.5, 55.6, 46.3, 43.9. HRMS
(EI-MS) calculated for C21H21N3O6 [M+H]+
412.1503, found 412.1507.
Chapter 3 | 85
5,7-Dimethoxy-2-(4-(3-nitrobenzyl)piperazin-1-yl)-4H-chromen-4-one (39).
White solid. Yield: 43 mg, 30%. Mp. 148−149°C. 1H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H),
8.18 – 8.13 (m, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.53 (t, J = 8.0 Hz, 1H), 6.34 (s, 2H), 5.40 (s, 1H),
3.92 (s, 3H), 3.85 (s, 3H), 3.69 (s, 2H), 3.55 – 3.44 (m, 4H), 2.69 – 2.55 (m, 4H). 13
C NMR (101
MHz, CDCl3) δ 177.4, 163.0, 161.1, 160.7, 157.4, 148.5, 139.4, 135.0, 129.4, 123.8, 122.7,
107.3, 95.8, 92.4, 88.6, 61.7, 56.4, 55.6, 52.0, 44.5. HRMS (EI-MS) calculated for C22H23N3O6
[M+H]+
426.1660, found 426.1663.
Methyl 4-((4-(5,7-dimethoxy-4-oxo-4H-chromen-2-yl)piperazin-1-yl)methyl)benzoate (40).
White solid. Yield: 62 mg, 42%. Mp.138−140°C. 1H NMR (300 MHz, CDCl3) δ 8.01 (d, J = 8.2
Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 6.36 (s, 2H), 5.42 (s, 1H), 3.91 (d, J = 1.6 Hz, 6H), 3.84 (s,
3H), 3.61 (s, 2H), 3.50 – 3.42 (m, 4H), 2.60 – 2.50 (m, 4H). 13
C NMR (75 MHz, CDCl3) δ 177.8,
167.0, 163.0, 161.2, 160.6, 157.3, 142.9, 129.8, 129.4, 129.0, 107.5, 95.8, 92.4, 88.4, 62.4, 56.5,
55.6, 52.2, 44.6. HRMS (EI-MS) calculated for C24H26N2O6 439.1864, found 439.1861.
5,7-Dimethoxy-2-((3-morpholinopropyl)amino)-4H-chromen-4-one (53).
Colorless sticky gum. Yield: 47 mg, 40%. 1H NMR (300 MHz, CD3OD) δ 6.50 (d, J = 2.3 Hz,
1H), 6.46 (d, J = 2.3 Hz, 1H), 5.21 (s, 1H), 3.87 (d, J = 4.0 Hz, 6H), 3.73 – 3.68 (m, 4H), 3.34 (s,
br, 2H), 2.52-2.47 (m, 6H), 1.97 – 1.73 (m, 2H). 13
C NMR (75 MHz, CD3OD) δ 165.1, 161.8,
158.7, 96.8, 93.9, 67.7, 57.2, 56.4, 56.4, 54.7, 40.7, 26.5. HRMS (EI-MS) calculated for
C18H24N2O5 349.1758, found 349.1761.
4-((4-(5,7-Dimethoxy-4-oxo-4H-chromen-2-yl)piperazin-1-yl)methyl)benzonitrile (41).
White solid. Yield: 40 mg, 31%. Mp. 185−187°C. 1H NMR (300 MHz, CDCl3) δ 7.58 (d, J = 8.2
Hz, 2H), 7.42 (d, J = 8.2 Hz, 2H), 6.31 – 6.22 (m, 2H), 5.38 (s, 1H), 3.86 (s, 3H), 3.79 (s, 3H),
3.56 (s, 2H), 3.45 – 3.32 (m, 4H), 2.54 – 2.41 (m, 4H). 13
C NMR (75 MHz, CDCl3) δ 177.8,
163.0, 161.2, 160.6, 157.4, 143.3, 132.3, 129.5, 118.8, 111.3, 107.3, 95.8, 92.4, 88.4, 62.2, 56.4,
55.6, 52.2, 44.6.HRMS (EI-MS) calculated for C23H23N3O4 [M+H]+
406.1761, found 406.1760.
5,7-Dimethoxy-2-(4-(2-nitrobenzyl)piperazin-1-yl)-4H-chromen-4-one (42).
Chapter 3 | 86
White solid. Yield: 47 mg, 33%. Mp. 164−166°C. 1H NMR (300 MHz, CDCl3) δ 7.75 (d, J = 7.9
Hz, 1H), 7.49 (d, J = 4.0 Hz, 2H), 7.37 (td, J = 8.6, 3.9 Hz, 1H), 6.27-6.24 (m, 2H), 5.32 (s, 1H),
3.84 (s, 3H), 3.78 (s, 3H), 3.40 – 3.26 (m, 4H), 2.53 – 2.39 (m, 4H). 13
C NMR (75 MHz, CDCl3)
δ 177.7, 163.0, 161.1, 160.5, 157.3, 149.8, 132.9, 132.5, 131.0, 128.4, 124.6, 107.2, 95.8, 92.4,
88.3, 60.4, 59.0, 56.4, 55.6, 52.1, 44.6. HRMS (EI-MS) calculated for C22H23N3O6 [M+H]+
426.1660, found 426.1663.
5,7-Dimethoxy-2-(4-(4-(methylsulfonyl)benzyl)piperazin-1-yl)-4H-chromen-4-one (43).
White solid. Yield: 45 mg, 29%. Mp. 188−191°C.1H NMR (300 MHz, CDCl3) δ 7.91 (d, J = 8.3
Hz, 2H), 7.56 (d, J = 8.3 Hz, 2H), 6.33 (t, J = 2.3 Hz, 2H), 5.43 (s, 1H), 3.90 (s, 3H), 3.84 (s, 3H),
3.63 (s, 2H), 3.46 (t, J = 6.0 Hz, 4H), 3.06 (s, 3H), 2.55 (t, J = 6.0 Hz, 4H). 13
C NMR (75 MHz,
CDCl3) δ 176.8, 162.0, 160.1, 159.6, 156.3, 143.2, 138.5, 128.7, 126.5, 106.2, 94.8, 91.3, 87.3,
61.0, 55.3, 54.6, 51.1, 43.5. HRMS (EI-MS) calculated for C23H26N2O6S [M+H]+
459.1584,
found 459.1585.
5,7-Dimethoxy-2-(4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)-4H-chromen-4-one (44).
White solid. Yield: 41 mg, 27%. Mp. 180−183°C. 1H NMR (300 MHz, CDCl3) δ 7.58 (d, J = 8.1
Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 6.47 – 6.15 (m, 2H), 5.39 (s, 1H), 3.90 (s, 3H), 3.83 (s, 3H),
3.60 (s, 2H), 3.45 (d, J = 3.0 Hz, 4H), 2.54 (t, J = 6.0 Hz, 4H). 13
C NMR (75 MHz, CDCl3) δ
177.8, 163.0, 161.2, 160.6, 157.4, 141.7, 129.5, 129.2, 126.0, 125.3, 107.3, 95.8, 92.4, 88.3, 77.5,
77.1, 76.7, 62.2, 56.4, 55.6, 52.1, 44.6. HRMS (EI-MS) calculated for C23H24F3N2O4 [M+H]+
449.1683, found 499.1688.
5,7-Dimethoxy-2-(4-(4-((trifluoromethyl)thio)benzyl)piperazin-1-yl)-4H-chromen-4-one (45).
White solid. Yield: 34 mg, 21%. Mp. 192−195°C. 1
H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 8.0
Hz, 2H), 7.40 (d, J = 8.1 Hz, 2H), 6.32 (s, 2H), 5.40 (s, 1H), 3.91 (s, 3H), 3.84 (s, 3H), 3.58 (s,
2H), 3.46 (t, J = 4.0 Hz, 4H), 2.55 (t, J = 4.0 Hz, 4H). 13
C NMR (100 MHz, CDCl3) δ 176.7,
162.0, 160.2, 159.6, 156.4, 139.9, 135.4, 130.1, 128.9, 127.1, 106.3, 94.8, 91.4, 87.4, 61.1, 55.4,
54.6, 51.1, 43.6. HRMS (EI-MS) calculated for C23H24F3N2O4S [M+H]+
481.1403, found
481.1399.
Chapter 3 | 87
5,7-Dimethoxy-2-(4-(4-((trifluoromethyl)sulfonyl)benzyl)piperazin-1-yl)-4H-chromen-4-one (46).
White solid. Yield: 29 mg, 17%. Mp. 168−172 °C. 1H NMR (400 MHz, CDCl3) δ 8.01 (d, J =
8.2 Hz, 2H), 7.68 (d, J = 8.3 Hz, 2H), 6.33 (s, 2H), 5.40 (s, 1H), 3.91 (s, 3H), 3.85 (s, 3H), 3.69
(s, J = 10.6 Hz, 2H), 3.40 – 3.54 (m, 4H), 2.47 – 2.61 (m, 4H). 13
C NMR (101 MHz, CDCl3) δ
177.7, 163.1, 161.2, 160.7, 157.4, 148.0, 131.0, 130.0, 107.4, 95.8, 92.4, 88.5, 62.0, 56.4, 55.6,
52.3, 44.6. HRMS (EI-MS) calculated for C23H24F3N2O4S [M+H]+ 481.1403, found 481.1399.
2-(4-(4-Aminobenzyl)piperazin-1-yl)-5,7-dimethoxy-4H-chromen-4-one (47).
Iron powder (236 mg, 4.2 mmol, 10.0 equiv.) and conc. HCl (ca. 2 mg) were added to a solution
of 32 (180 mg, 0.42 mmol, 1.0 equiv.) in 9 mL EtOH and 2.25 mL water. The mixture was
heated to reflux for 90 min. EtOAc was added to the mixture and dried with Na2SO4. After
filtration and evaporation of the solvent, the residue was purified by chromatography (EtOAc :
MeOH = 10:1) to afford pale yellow solid. Yield: 132 mg, 80%. Mp. 174−176°C. 1H NMR (400
MHz, CDCl3) δ 7.09 (d, J = 8.3 Hz, 2H), 6.64 (dd, J = 8.6, 2.1 Hz, 2H), 6.32-6.30 (m, 2H), 5.31
(s, 1H), 3.89 (s, 3H), 3.83 (s, 3H), 3.45 – 3.41(m, 6H), 2.57 – 2.45 (m, 4H). 13
C NMR (101 MHz,
CDCl3) δ 177.5, 162.8, 161.1, 160.5, 157.3, 145.8, 130.4, 114.9, 107.4, 95.7, 92.3, 88.3, 62.3,
56.3, 55.5, 51.8, 44.5. HRMS (EI-MS) calculated for C22H25N3O4 [M+H]+
396.1918, found
396.1923.
N-(4-((4-(5,7-Dimethoxy-4-oxo-4H-chromen-2-yl)piperazin-1-yl)methyl)phenyl)acetamide (48).
To an ice-cold of 47 (132 mg, 0.33mmol, 1.0 equiv.) in DCM (10mL) was added Et3N (100 mg,
0.99mmol, 3.0 equiv.) dropwise and the reaction mixture was stirred at the same temperature for
15 min. Acetyl chloride (39 mg, 0.495mmol, 1.5 equiv.) was then added and the reaction was
stirred at rt for 1 h. After completion of the reaction, 10 mL water was added and the mixture
extracted with DCM (20 mL × 3). The combined organic phase was washed with brine (20 mL ×
3), dried over anhydrous Na2SO4. After evaporation of the solvent, the residue was purified by
flash chromatography (EtOAc: MeOH=10:1) to give a white solid. Yield: 124 mg, 86%. Mp.
176−178°C. 1H NMR (300 MHz, CDCl3) δ 8.74 (s, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.4
Hz, 2H), 6.33 (d, J = 2.3 Hz, 1H), 6.29 (d, J = 2.3 Hz, 1H), 5.29 (s, 1H), 3.83 (s, 6H), 3.48 (s,
2H), 3.44 – 3.33 (m, 4H), 2.58 – 2.40 (m, 4H), 2.14 (s, 3H). 13
C NMR (75 MHz, CDCl3) δ 177.7,
169.1, 163.0, 161.2, 160.5, 157.4, 137.9, 132.5, 129.6, 119.9, 107.2, 95.8, 92.4, 88.1, 62.3, 56.2,
Chapter 3 | 88
55.7, 51.9, 44.5, 24.4. HRMS (EI-MS) calculated for C24H27N3O5 [M+H]+ 438.2023, found
438.2028.
5,7-Dimethoxy-2-(4-methylpiperazin-1-yl)-4H-chromen-4-one (49).
Pale yellow solid. Yield: 39 mg, 38%. Mp.117−118°C. 1H NMR (300 MHz, CDCl3) δ 6.27 (m,
2H), 5.28 (s, 1H), 3.85 (s, 3H), 3.79 (s, 3H), 3.43 – 3.34 (m, 4H), 2.49 – 2.39 (m, 4H), 2.28 (s,
3H). 13
C NMR (75 MHz, CDCl3) δ 177.7, 162.9, 161.2, 160.6, 157.4, 107.4, 95.8, 92.3, 88.4,
56.4, 55.6, 54.1, 46.1, 44.5. HRMS (EI-MS) calculated for C16H20N2O4 [M+H]+
305.1496, found
305.1499.
5,7-Dimethoxy-2-(4-(4-nitrobenzoyl)piperazin-1-yl)-4H-chromen-4-one (50).
White solid. Yield: 43 mg, 29%. Mp. 248−250°C. 1H NMR (300 MHz, CDCl3) δ 8.30 (d, J = 8.6
Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 6.33 (s, 2H), 5.36 (s, 1H), 4.03-3.78 (t, J = 19.2 Hz, 8H), 3.66-
3.26 (m, 6H). 13
C NMR (75 MHz, CDCl3) δ 177.5, 168.2, 163.2, 160.7, 160.7, 157.4, 148.7,
141.0, 128.2, 124.1, 107.3, 96.0, 92.4, 89.3, 56.5, 55.7, 53.5.HRMS (EI-MS) calculated for
C22H21N3O7 [M+H]+
440.1452, found 440.1450.
2-(4-(Benzo[c][1,2,5]oxadiazol-5-ylmethyl)piperazin-1-yl)-5,7-dimethoxy-4H-chromen-4-
one (51).
White solid. Yield: 35 mg, 25%. Mp. 204−206°C. 1
H NMR (300 MHz, CDCl3) δ 7.80 (d, J = 9.3
Hz, 1H), 7.72 (s, 1H), 7.50 (dd, J = 9.3, 1.1 Hz, 1H), 6.51 – 6.14 (m, 2H), 5.36 (s, 1H), 3.91 (s,
3H), 3.84 (s, 3H), 3.63 (s, 2H), 3.47 (t, J = 3.0 Hz, 4H), 2.60 (t, J = 6.0 Hz, 4H). 13
C NMR (75
MHz, CDCl3) δ 177.7, 163.0, 161.1, 160.6, 157.4, 149.3, 149.0, 142.2, 133.2, 116.5, 114.5,
107.4, 95.8, 92.4, 88.5, 62.4, 56.4, 55.6, 52.2, 44.6. HRMS (EI-MS) calculated for C22H22N4O5
[M+H]+
423.1663, found 423.1667.
2-((3,4-Dimethoxyphenethyl)amino)-5,7-dimethoxy-4H-chromen-4-one (52).
White solid. Yield: 52 mg, 40%. Mp.80−82°C. 1H NMR (300 MHz, CDCl3) δ 6.81 – 6.65 (m,
3H), 6.26 (d, J = 2.4 Hz, 1H), 6.22(d, J = 2.4 Hz, 1H), 5.30 (s, 1H), 3.86 (s, 3H), 3.83 (d, J = 1.8
Chapter 3 | 89
Hz, 6H), 3.79 (s, 3H), 3.39 (s, 2H), 2.86 (t, J = 7.0 Hz, 2H). 13
C NMR (75 MHz, CDCl3) δ 177.4,
162.8, 161.6, 160.5, 157.3, 149.1, 147.9, 130.5, 120.7, 111.9, 111.4, 107.3, 95.7, 92.5, 86.2, 56.4,
55.9, 55.6, 43.0, 34.5, 31.6. HRMS (EI-MS) calculated for C21H23NO6 [M+H]+
386.1603, found
386.1606.
General synthetic procedure for final products 62-67.
A 50 mL schlenk flask was charged with 59-61 (1.0 equiv.), DIPEA (10.0 equiv.) and HBTU
(3.0 equiv.) under nitrogen atmosphere. After dissolving in 10 mL of dried DCM, N-subtituted
piperazines 11-13, 21 and 29 (1.0 equiv.) was slowly added at 0°C. Then the mixture was stirred
for 24 h at room temperature. The organic phase was washed with water and brine and dried over
anhydrous Na2SO4. After purification with flash chromatography column (EtOAc: MeOH =
30:1), the final products 62-67 were obtained.
5,7-Dimethoxy-3-(4-(3-nitrophenethyl)piperazine-1-carbonyl)-4H-chromen-4-one (62).
White solid. Yield: 159 mg, 85%. Mp.169−171°C. 1H NMR (300 MHz, DMSO-d6) δ 8.15 (d, J =
8.6 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H), 6.72 (d, J = 2.2 Hz, 1H), 6.54 (d, J = 2.1 Hz, 1H), 6.24 (s,
1H), 3.85 (d, J = 13.8 Hz, 6H), 3.54 (d, J = 23.6 Hz, 4H), 2.90 (t, J = 7.2 Hz, 2H), 2.63 (t, J = 7.3
Hz, 2H). 13
C NMR (75 MHz, DMSO-d6) δ 32.0, 56.0, 56.1, 58.0, 93.3, 96.5, 108.6, 111.9, 123.2,
129.9, 145.8, 149.0, 154.9, 158.8, 160.3, 164.0, 174.7. HRMS (EI-MS) calculated for
C24H25N3O7 [M+H]+ 468.1765, found 468.1173.
5,7-Dimethoxy-2-(4-(4-nitrobenzyl)piperazine-1-carbonyl)-4H-chromen-4-one (63).
White solid. Yield: 123 mg, 68%. Mp. 191−192°C. 1H NMR (300 MHz, CDCl3) δ 8.18 (d, J =
8.7 Hz, 2H), 7.51 (d, J = 8.7 Hz, 2H), 6.44 (d, J = 2.3 Hz, 1H), 6.36 (d, J = 2.3 Hz, 1H), 6.32 (s,
1H), 3.92 (s, 3H), 3.86 (s, 3H), 3.76 (s, 2H), 3.63 (s, 2H), 3.59 (s, 2H), 2.51 (s, 4H). 13
C NMR
(75 MHz, CDCl3) δ 176.3, 164.5, 161.0, 161.0, 159.4, 154.7, 147.4, 145.4, 129.5, 123.7, 113.4,
109.6, 96.7, 92.9, 61.8, 56.5, 55.9. HRMS (EI-MS) calculated for C23H23N3O7 [M+H]+ 454.1609,
found 454.1622.
5-Hydroxy-7-methoxy-2-(4-(2-nitrobenzyl)piperazine-1-carbonyl)-4H-chromen-4-one (64).
White solid. Yield: 100 mg, 54%. Mp. 144−146°C. 1H NMR (300 MHz, CDCl3) δ 12.36 (s, 1H),
7.82 (d, J = 7.9 Hz, 1H), 7.61 – 7.48 (m, 2H), 7.47 – 7.38 (m, 1H), 6.38 (q, J = 2.1 Hz, 3H), 3.86
Chapter 3 | 90
(s, 5H), 3.70 (s, 2H), 3.49 (s, 2H), 2.50 (s, 4H). 13
C NMR (75 MHz, CDCl3) δ 181.6, 166.1,
162.2, 160.4, 158.2, 157.2, 149.9, 144.1, 132.5, 131.0, 128.5, 124.7, 110.4, 106.1, 98.8, 92.9,
58.9, 55.9, 53.2, 52.2, 42.4. HRMS (EI-MS) calculated for C22H21N3O7 [M+H]+ 440.1452, found
440.1450.
5-Hydroxy-7-methoxy-2-(4-(4-nitrobenzyl)piperazine-1-carbonyl)-4H-chromen-4-one (65).
White solid. Yield: 149 mg, 60%. Mp.184−186°C. 1H NMR (400 MHz, CDCl3) δ 12.33 (s, 1H),
8.18 (d, J = 8.7 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H), 6.39 (d, J = 2.2 Hz, 1H), 6.37 (s, 1H), 6.36 (d,
J = 2.2 Hz, 1H), 3.85 (s, 3H), 3.67 (d, J = 8.4 Hz, 4H), 3.65 (s, 2H), 2.53 (d, J = 19.6 Hz, 4H).
13C NMR (100 MHz, CDCl3) δ 181.6, 166.1, 162.3, 160.5, 158.1, 157.2, 147.4, 145.3, 129.5,
123.7, 110.5, 106.1, 98.7, 93.1, 61.8, 55.9, 53.2, 52.4, 47.0, 42.4. HRMS (EI-MS) calculated for
C22H21N3O7 [M+H]+ 440.1452, found 440.1450.
5-(Benzyloxy)-2-(4-benzylpiperazine-1-carbonyl)-4H-chromen-4-one (66).
White solid. Yield: 132 mg, 86%. Mp: 169−171°C. 1H NMR (300 MHz, CDCl3) δ 7.59 (d, J =
7.3 Hz, 2H), 7.53 (t, J = 8.4 Hz, 1H), 7.40 (t, J = 7.4 Hz, 2H), 7.36 – 7.27 (m, 6H), 7.06 – 6.99
(m, 1H), 6.87 (d, J = 8.1 Hz, 1H), 6.41 (s, 1H), 5.28 (s, 2H), 3.77 (s, 2H), 3.58 (d, J = 12.4 Hz,
4H), 2.52 (d, J = 19.8 Hz, 4H). 13
C NMR (101 MHz, CDCl3) δ 176.8, 160.8, 158.6, 157.7, 155.6,
137.2, 136.2, 134.2, 129.0, 128.5, 128.3, 127.7, 127.4, 126.6, 115.4, 113.3, 110.4, 108.9, 70.8,
62.6, 53.0, 52.3. HRMS (EI-MS) calculated for C26H26N2O4 [M+H]+ 455.1965, found 455.1962.
5-(Benzyloxy)-N-(3,4-dimethoxyphenethyl)-4-oxo-4H-chromene-2-carboxamide (67).
Pale yellow solid. Yield: 123 mg, 80%. Mp. 186−188°C. 1
H NMR (300 MHz, CDCl3) δ 7.63 –
7.48 (m, 3H), 7.43 – 7.22 (m, 3H), 6.98 – 6.71 (m, 6H), 5.26 (s, 2H), 3.87 (d, J = 4.2 Hz, 6H),
3.70 (q, J = 6.8 Hz, 2H), 2.90 (t, J = 7.0 Hz, 2H). 13
C NMR (75 MHz, CDCl3) δ 177.6, 159.3,
158.8, 157.2, 152.6, 149.2, 147.9, 136.2, 134.4, 130.8, 128.7, 127.8, 126.6, 120.7, 115.5, 113.7,
111.9, 111.4, 110.0, 108.9, 70.9, 56.0, 55.9, 41.1, 35.1. The spectroscopic data are in accordance
with literature.[39]
Biology
Chapter 3 | 91
Calcein-AM and Hoechst 33342 assay:
Solutions
Loading buffer was made of 120 mM NaCl, 5 mM KCl, 2 mM MgCl2 6H2O, 1.5 mM
CaCl2·2H2O, 25 mM HEPES, 10 mM glucose, the pH was adjusted to 7.4.
Phosphate buffered saline (PBS) was made of 8 g/L NaCl, 1 g/L Na2HPO4, 0.2 g/L KCl, 0,2
g/L KH2PO4 and NaH2PO4·H2O, finally the pH was adjusted to 7.4.
4% Paraformaldehyde (PFA) solution in PBS was obtained by stirring 4 g PFA per 100 g total
solution while heating on a magnetic stirrer for approximately 30 min. If not otherwise stated,
chemicals (p.a. quality) were purchased from Merck (Darmstadt, Germany). Purified water
(Milli-Q system, Millipore, Eschborn, Germany) was used.
Chemicals used for cellular assays
Calcein-AM and Hoechst 33342 were from Biotium (Hayward, CA, USA). Calcein-AM was
dissolved in DMSO (Merck, Darmstadt, Germany) at a concentration of 1 mM, whereas as
solution of Hoechst 33342 (0.8 mM) was prepared in Millipore water. Pluronic® F127 was
obtained from Sigma (Munich, Germany) and dissolved to achieve a final concentration of 20%
(m/m) in DMSO. Reversan and fumitremorgin C (FTC) were from Merck (Darmstadt, Germany),
dissolved in DMSO and diluted to achieve a concentration of 3 mM and 1 mM, respectively.
Vinblastine, topotecan and etoposide were from Sigma (Munich, Germany) and dissolved in 70%
ethanol. Tariquidar was synthesised according to literature [41]
with minor modifications,[42]
compounds 66 and 67 were synthesised as reference compounds according to a published
protocol.[11]
Work solutions of vinblastine and topotecan at a concentration of 0.1 mM were
stored at 4°C, and all stock solution at −20°C.
Cell culture conditions
Human Kb-V1 cells, a subclone of human Kb cells (ATCC® CCL-17™), overexpressing
ABCB1 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma, Munich,
Germany) supplemented with 3.7 g/L NaHCO3 (Merck, Darmstadt, Germany) and 110 mg/L
sodium pyruvate (Serva, Heidelberg, Germany) containing 10% FCS (Biochrom, Berlin,
Chapter 3 | 92
Germany) and vinblastine at a final concentration of 330 nM to maintain the ABCB1 transporter
expression.[42]
The human breast adenocarcinoma cell line MCF-7/Topo (ABCG2 overexpressing subclone of
MCF-7 cells, ATCC® HTB-22™) were incubate in Eagle’s Minimum Essential Medium
containing 2.2 g/L NaHCO3 (Merck, Darmstadt, Germany) and 110 mg/L sodium pyruvate
(Serva, Heidelberg, Germany) and 10% FCS (Biochrom) and 550 nM topotecan to maintain the
ABCG2 transport overexpression. The ABCG2 overexpression was described previously.[8b]
The MDCKII-MRP1 cell line (Madin-Darby Canine Kidney, strain II; a canine epithelial cell
line; ATCC® CRL-2936™ transfected with the human ABCC1 transporter [43]
) was a kind gift
from Prof. Dr. P. Borst from Netherland Cancer Institute (Amsterdam, NL). These cells were
maintained in DMEM supplemented with 10% FCS (Biochrom).[16]
All cells were cultured in a water-saturated atmosphere (5% CO2) at 37°C in cell culture flasks
purchased from Sarstedt (Nümbrecht, Germany). Subculturing of cells was performed every 3-7
days after trypsinization (0.05% trypsin / 0.02% EDTA or 0.1% trypsin / 0.04% EDTA, GE
Healthcare (PAA Laboratories, Pasching, Austria). Additionally the cells were routinely
monitored for mycoplasma contamination by PCR (Venor® GeM, Minerva Biolabs, Berlin,
Germany) and only mycoplasma free cultures were used.
Modulation of ABCB1:
ABCB1 modulation was determined in a microplate assay (calcein-AM assay) as described.[44]
Modulation of ABCG2:
ABCG2 modulation was determined with the Hoechst 33342 microplate assay as described.[44]
Chapter 3 | 93
Modulation of ABCC1:
ABCC1 modulation was determined with the calcein-AM microplate assay by analogy with the
procedure established for ABCB1.[16]
Briefly, 3-5 days after passaging, the ABCC1
overexpressing MDCKII-MRP1 cells were trypsinized and resuspended in culture medium at
25°C. The cells were seeded into flat-bottomed 96-well plates at a density of 20000 – 25000 cells
per well. The next day, cells were washed with loading buffer in order to remove unspecific
serum esterases. Afterwards, cells were incubated with loading suspension (loading buffer, 5
mg/mL BSA, 1.25 μL/mL pluronic F127 (20% in DMSO) containing 0.5 μM of calcein-AM and
vehicle or the test compound at increasing concentrations (10 nM – 100 µM) for 60 min (37°C /
5% CO2). Reversan at a final concentration of 30 µM was used as reference compound; the
response obtained under these conditions was defined as 100% inhibition of calcein-AM efflux.
In general, test compounds were investigated from two to four independent experiments
performed in triplicate, in case of controls in sextuplicate. Subsequently, the loading suspension
was discarded and cells were fixed under light protection using 100 µL of a PFA solution (4 % in
PBS) for 20 min. After three washing circles with 150 µL of loading buffer, fixed cells were
overlaid with 100 µL of loading buffer and relative fluorescence intensities were determined at a
GENios Pro microplate reader (Tecan Deutschland GmbH, Crailsheim, Germany). Measurement
mode: fluorescence top; excitation filter: 485/20 nm; emission filter: 535/25 nm; number of reads:
10; integration time: 40 µs; lag time: 0 µs; mirror selection: Dichroic 3; plate definition file:
GRE96ft.pdf; multiple reads per well (Circle): 3x3; time between move and flash: 100 ms. On
each plate, the optimal gain was calculated by determination of the fluorescence in absence and
presence of the reference compound reversan (30 µM). All values were corrected by subtracting
the fluorescence intensity in the absence of ABCC1 modulator (DMSO control value), and the
maximal response was referred to the signal obtained using 30 µM reversan (100%). IC50 values
were calculated using SigmaPlot 11.0, “four parameter logistic curve” fitting. Errors are
expressed as standard error of the mean (SEM)
Determination of the stability of compound 51 in mouse plasma
Chapter 3 | 94
Compound stability in mouse plasma was determined according to a previously established
procedure.[8d]
The blood from NMRI (nu/nu) mice was collected by heart puncture in deep
anesthesia using heparin-coated syringes. Samples were immediately centrifuged for 7 min at
4500 g (Eppendorf centrifuge 5415R, Eppendorf, Hamburg, Germany). The test compound was
dissolved in DMSO at a concentration of 7 mM. A 1:50 dilution of substance with mouse plasma
was prepared in 1.5 mL polypropylene reaction vessels (Eppendorf, Hamburg, Germany). The
samples were shortly vortexed and promptly incubated at 37°C. Aliquots were obtained at
different incubation times. The samples were deproteinated by mixing with two parts of ice-cold
acetonitrile (MeCN). For quantitative precipitation, the samples were vortexed and stored at 4°C
for 30 min. Afterwards the samples were centrifuged for 5 min at 14000 g (Eppendorf centrifuge
5415 R) and the supernatants were transferred into new reaction vessels. The samples were
diluted (1:1) with MeCN and stored at −80°C until the HPLC analysis.
RP-HPLC analysis was performed with a Eurosphere-100 C18 column (250 x 4 mm, 5 µm,
Knauer, Berlin, Germany), maintained to 30°C, on a Merck Hitachi system consisting of an AS-
2000A , a L-6200-A pump, a L-400A UV-VIS detector. UV-detection was done at 220 nm. The
samples were thawed at room temperature and 100 µL were injected. Mixtures of MeCN (A) and
0.05 % aq. TFA (B) were used as mobile phase. The applied gradient was 0 to 30 min (A/B):
10/90 to 80/20 in 30 min.
Chemosensitivity Assays
The assays were performed according to an established protocol [45]
with minor modifications.[8d]
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Chapter 3 | 98
[45] G. Bernhardt, H. Reile, H. Birnböck, T. Spruß, H. Schönenberger, J. Cancer Res. Clin. Oncol.
1992, 118, 35-43.
Chapter 3 | 99
1H and
13C NMR spectra of selected final compounds
1H and
13C NMR spectra for 5,7-dimethoxy-2-(4-(4-((trifluoromethyl)sulfonyl)benzyl)piperazin-
1-yl)-4H-chromen-4-one (46) (400 MHz, CDCl3)
Chapter 3 | 100
1H and
13C NMR spectra for 2-(4-(benzo[c][1,2,5]oxadiazol-5-ylmethyl)piperazin-1-yl)-5,7-
dimethoxy-4H-chromen-4-one (51) (300 MHz, CDCl3)
Chapter 3 | 101
1H and
13C NMR spectra for 5,7-dimethoxy-2-(4-(4-nitrobenzyl)piperazine-1-carbonyl)-4H-
chromen-4-one (62) (300 MHz, DMSO-d6)
Chapter 4 | 102
The data discussed in this chapter have been published as part of the following paper:
S. Bauer, C. Ochoa-Puentes, Q. Sun, M. Bause, G. Bernhardt, B. König, and A.Buschauer.
ChemMedChem, 2013, 11, 1773-1778.
Author contributions:
Q.Sun synthesized and characterized quinoline derivatives.
Chapter 4
Quinoline carboxamide-type ABCG2 modulators: quinoline moiety as anilide
replacement
Abstract
A new series of quinoline analogues targeting breast cancer resistance protein derived from
tariquidar was synthesised and tested in ABCB1, ABCG2 and ABCC1 assay. The replacement of
anilide core by quinoline moiety increased the stability compared with parent compounds UR-
ME22-1, UR-COP78 but gave less potent compounds than indole compound UR-COP-25g. The
quinoline analogues were less water soluble than UR-ME22-1, UR-COP78 and UR-COP-25g
due to coplanar structures. The introduction of amine groups on the tetrahydroisoquinoline
moiety increased the water solubility to some extent.
Keywords
ABC transporters; breast cancer resistance proteins; inhibitors; quinoline; syntheses
Chapter 4 | 103
Introduction
The ATP-driven drug efflux transporters ABCB1 (p-glycoprotein, p-gp), ABCC1 (MRP1), and
ABCG2 (breast cancer resistance protein, BCRP) play an important role in multidrug resistance
(MDR) of cancer.[1]
In addition, the chemotherapy of malignant CNS tumors is compromised
due to the expression of ABC proteins at the blood-brain barrier, restricting the access of many
potent cytostatics to the brain, in the case that these compounds are substrates of the respective
pumps. The coadministration of such cytostatics with inhibitors of efflux transporters represents
an attractive strategy to overcome the blood-brain barrier and to improve chemotherapy of
malignancies in the CNS, as demonstrated in a proof-of-concept study for ABCB1.[2]
It is
expected that this concept is also applicable to ABCG2, provided that appropriate inhibitors are
available.
Recently, we identified selective ABCG2 inhibitors among 3-
(quinolinecarbonylamino)benzanilides (Figure 1).[3]
With an IC50 value of 65 nm and a maximal
inhibitory effect of 63% (Hoechst 333342 assay, MCF7-Topo cells), UR-ME22-1 turned out to
be more potent but less efficient compared to the reference compound fumitremorgin C (FTC).
The incorporation of a triethylenglycol ether group at the tetrahydroisoquinoline moiety gave
compound UR-COP78, which is comparable to UR-ME22-1 in potency but produces a higher
maximal response of 88%, indicating that low water solubility was an efficacy-limiting factor.[3b]
Unfortunately, these compounds were unstable in mouse plasma due to complete enzymatic
cleavage of the benzamide bond within 30 min, giving the corresponding phenethyl
tetrahydroisoquinoline fragment.[4]
Aiming at more stable ABCG2 modulators, we replaced the
labile benzanilide core structure according to a bioisosteric approach. A biphenylyl moiety was
tolerated, but potency and selectivity were reduced compared to the parent compounds.[4a]
Replacement of biphenyly moiety with indole moiety largely increased the activity to IC50 value
= 59 ± 11nm with maximal inhibitory effect of 101%, besides it was stable in mouse plasma for
24 hours.[5]
For better understanding the structure-activity relationship on this class of
compounds, the modification of fragment C and D (Figure. 1) has been intensively studied in
our group. Here we report on the replacement of the indole core on fragment B by quinolyl
Chapter 4 | 104
moieties and introduction of amine on fragment A to increase water solubility rather than
triethylenglycol ether group.
Figure 1. The selective ABCG2 modulators UR-ME22-1, UR-COP78 and UR-COP-25g and the general
structures of the title compounds.
Results and Discussion
Synthesis
As shown in Figure 1, our target compounds constitute four fragments: the
tetrahydroisoquinoline moiety (fragment A), the quinoline motif (fragment B), the methyl
Chapter 4 | 105
aminobenzoate moiety (fragment C) and the quinoline-2-carbonyl core (fragment D) (dashed
ovals, Figure 1). At the beginning, we focused on the synthesis of quinoline core. Cottet et al.[6]
have developed an ingenious synthesis of the precursor 3 by high-temperature Lewis acid-
catalysed cyclization of the N-arylcinnamamide 2 in a reaction that formally generates benzene
as a leaving group (Scheme 1). Following this route, 4-methylaniline 1 was acylated with
cinnamoyl chloride to afford 2 in high yield. Reaction of 2 with AlCl3 at 125°C led to a 41%
isolated yield of the quinolin-2-one 3. Consequently, bromination 3 with POBr3 gave 2-Bromo-
6-methylquinoline 4 in 61% yields.
Scheme 1. Synthesis of compound 2-Bromo-6-methylquinoline 4. Reagents and conditions: (a) E-
PhCHCHCOCl, K2CO3, water, acetone, 0°C, 2 h; (b) AlCl3, PhCl, 125°C, 24 h; (c) POBr3, 140°C, 3 h.
Water solubility plays a very important role in drug absorption and affects the bioavailability of
drugs.[7]
To increase water solubility, triethylene glycol was introduced to tetrahydroisoquinoline
moiety (Scheme 2), After efficient cleavage of methoxy groups with HBr/CH3COOH, free
secondary amine in the molecule was protected by boc reagent. The triethylene glycol
monomethyl ether was activated as tosylate and then used to form the ether 8 under basic
conditions. The deprotection of intermediate 8 by trifluoro acetic acid (TFA) gave free amine as
triflate salt 9 in quantitative yield.
Substitution of two triethylene glycol chains largely increased the solubility of the compounds,
but meanwhile decreased the selectivity between ABCB1 and ABCG2 transporters, while one
triethylene glycol substitution did not affect the selectivity of the compound (Table 1). Another
strategy to increase the water solubility is to introduce polar tertiary amine group into the
molecule. Therefore we synthesised a series of amine substituted tetrahydroisoquinoline moiety.
Chapter 4 | 106
The synthesis of mono-substituted tetrahydroisoquinoline moiety has been reported (Scheme 3)
[8].
Scheme 2. Synthesis of tetrahydroisoquinoline moiety 9. Reagents and conditions: (a) HBr/CH3COOH,
reflux; (b) Boc2O, Et3N, DCM, 0°C; (c) 2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate,
KOH, THF, reflux; (d) TFA, DCM, r.t.
Scheme 3. Synthesis of tetrahydroisoquinoline moiety 22-27. Reagents and conditions: (a) 2,2-
diethoxyethanamine, EtOH, reflux, 5 h; (b) Pt/H2, 40bar, 3 ds; (c) 6N HCl, rt, 3 ds; (d) Pd/C, H2, 40 bar, 1
d; (e) Boc2O, Et3N, DCM, overnight; (f) 2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate or
aliphatic amines, reflux, overnight; (g) TFA, DCM, r.t.
Chapter 4 | 107
Condensation of isovanillin 10 and aminoacetaldehyde diethyl acetal in ethanol gave imine 11
which was used directly in the next step without any workup. After reduction to amine 12, it was
treated with dilute hydrochloric acid and followed by hydrogenation with Pd/H2 to yield the
asymmetrically substituted tetrahydroisoquinoline with quantitative yield. The amine can be
selectively protected with di-tert-butyl dicarbonate due to its higher reactivity than phenol.
Followed by alkylation and deprotection, mono-substituted tetrahydroisoquinoline moieties 22-
27 were obtained as triflates. [9]
Palladium catalysed borylation of arylbromide gave fluorescent boronic ester 29 in 90% yield.
The Suzuki coupling of boronic ester 29 with 2-bromo-6-methyl quinoline 4 to 2-bromo-6-
methyl quinolone 30 followed by the acylation with quinoline-2-carbonyl chloride gave
compound 31 in good yield. NBS was added in three portions to give one-bromonated product
32, which was used directly to the next step after work-up. The final compounds 33a-h were
generated by nucleophilic substitution reaction (Scheme 4).
Chapter 4 | 108
Scheme 4. Synthesis of title compounds 33a-h. Reagents and conditions: (a) Pd(dppf)Cl2, KOAc, DMSO,
80°C, overnight; (b) 2-bromo-6-methyl quinoline 4, Pd(PPh3)4, K3PO4, THF, 80°C, overnight ； (c)
quinoline-2-carbonyl chloride, TEA, DCM, 40°C, overnight; (d) NBS, benzoyl peroxide, CCl4, 80°C, 9 h;
(e) tetrahydroisoquinolines 22-27, DIPEA, CH3CN, 80°C, overnight.
Biological evaluation
Inhibition of the ABCB1 and ABCG2 transporter
The synthesised compounds 33a-h and the reference compounds fumitremorgin C, Ko143[10]
and
tariquidar were investigated for inhibition of ABCB1 and ABCG2 in a calcein-AM[11]
and a
Chapter 4 | 109
Hoechst 33342 microplate assay[4b]
using ABCB1-overexpressing Kb-V1 and ABCG2-
overexpressing MCF-7/Topo cells. The data are summarized in Table 1.
Table 1. Effect of 33a-h and reference compounds on the transport activity of ABCG2 and ABCB1
Compound ABCG2[a]
ABCB1[b]
IC50 [nm][c]
Imax [%][d]
IC50 [nm][c]
Imax [%][e]
FTC 731 ± 92 100 n.d.
Ko143[f]
117 ± 53 103 ± 7 inactive[g]
Tariquidar[f]
526 ± 85 69 ± 5 223 ± 8 103 ± 2
UR-ME22-1[f]
65 ± 8 63 ± 2 >29000[h]
UR-COP78[i]
130 ± 29 88 ± 3
>50000
UR-COP-25g[j]
59 ± 11 101 ± 5
7300 ± 910 14 ± 2
33a 602 ± 44 60 ± 0 inactive
33b 536 ± 160 77 ± 1 2709 ± 158 41 ± 15
33c 1043 ± 53 107 ± 7 inactive
33d 851 ± 93 81 ± 10 inactive
33e 904 ± 45 58 ± 0 >10000
33f 1167 ± 214 61 ± 5 >10000
33g 1467 ± 568 70 ± 5 inactive
33h 1820 ± 861
75 ± 5 >10000
[a] Hoechst 33342 microplate assay using ABCG2-overexpressing MCF-7/Topo cells. [b] Calcein-AM
microplate assay (unless otherwise indicated) using ABCB1-overexpressing Kb-V1 cells. [c] Mean values ±
SEM from 2 to 3 independent experiments performed in triplicate or sextuplicate; n.d.: not determined.
[d] Maximal inhibitory effect (Imax) relative to the response to FTC at a concentration of 10 µm (100%
inhibition). [e] Imax expressed as percental inhibition relative to tariquidar at a concentration of 1 µm
(100%). [f] Ref.[4b]
[g] Inactive up to a concentration of 100 µm. [h] Data from flow cytometric calcein-AM
assay.[11]
[i] Ref.[3b]
. [j] Ref.[5]
As shown in Table 1, all the quinoline analogues exhibit higher IC50 value but lower the
maximal response compared with UR-COP-25g. The introduction of two triethylene glycol
Chapter 4 | 110
chains at the tetrahydroisoquinoline core (compound 33b) decreases the selectivity in ABCG2
transporter; whereas compound 33c with one triethylene glycol chain maintains the good
selectivity and showed higher maximal inhibitory effect than other compounds. The quinoline
analogues turned out to be hardly soluble which was precipitated in the pharmacological assay
leading to the big error of IC50 value. The poorer water solubility could be explained by the fact
that the strong conjugation of the nitrogen atom decreasing the possibility to form H-bonds and
the structure of the quinoline molecule is more planar than related analogues (Figure 2).
The modification of side chain at the tetrahydroisoquinoline core has slight effect on the ABCG2
activity. As shown in Table 2, the better water solubility tends to be accompanied with better
ABCG2 inhibitory effect. Quinoline 33d and 33h bearing morpholine substitution have lower
logP value showing a bit lower IC50 value than other amine substitution analogues (33f, 33g and
33h). Usually logP value of 5 is considered as an upper limit of desired lipophilicity for drug-like
molecule, but all the compounds (UR-COP-25g and 33a-h) went beyond the scope. The big
molecular weight of the compounds is also the factor decreasing hydrophilicity of the
compounds. Compound 33d which bears ethylmorpholine has almost the same logP value as
compound 33c and 33d. Interestingly, they also show the similar activity on ABCG2 transporter.
This suggests that ethylmorpholine can be used as a substitution of triethylene glycol chains to
improve the drug-like property of the compounds. None of the compounds show activity at the
ABCC1 transporter. Since quinoline compounds are inferior to UR-COP-25g, no future
investigation was performed on this series of compounds.
Chapter 4 | 111
Figure 2. The structure of quinoline analogue 33a (energy minimization by ChemBio 3D Ultra 13.0).
Table 2. Molecular weight and calculated logP value for indole analogue UR-COP-25g and quinoline
analogues 33a-h
Compound Molecular Weight logP[a]
UR-COP-25g 772.90 7.47
33a 638.72 7.84
33b 903.04 7.67
33c 770.88 7.71
33d 737.86 7.64
33e 751.88 7.70
33f 735.88 8.71
33g 751.90 8.77
33h 737.88 8.27 [a]
The partition coefficient (log p) of the quinoline compounds 33a-h and UR-COP-25g were calculated with Marvinsketch 5.10.1.
Chapter 4 | 112
Conclusions
A new class of quinoline analogues targeting breast cancer resistance protein derived from
tariquidar was synthesised and tested in ABCB1, ABCG2 and ABCC1 assay. All tested
compounds show weak or no inhibitory activity over ABCB1 and ABCC1 transporter but good
inhibitory activity towards ABCG2. However, these compounds have worse activities on
ABCG2 than reference indole compound UR-COP-25g. The replacement of triethylene glycol
chains with ethylmorpholine maintained the activity of the compounds and decreased molecular
weight. In future, our work should be focused on solving the solubility problem via structure-
activity study. Through optimization of the pharmacophore, the size of compounds can be
minimized and the hydrophilicity may largely be enhanced.
Experimental
Chemistry
1H,
13C and 2D NMR spectra were obtained at 298 K using a Bruker AVANCE 300 spectrometer
(operating at 300.13 MHz for 1H and 75.47 MHz for
13C), Bruker AVANCE 400 spectrometer (operating
at 400.13 MHz for 1H and 100.62 MHz for
13C) and Bruker AVANCE 600 spectrometer (operating at
600.25 MHz for 1H and 150.93 MHz for
13C) (Bruker, Karlsruhe, Germany). The spectra were
obtained using chloroform-d (99.8%, Deutero GmbH) or methanol-d4 (99.8%, Deutero GmbH)
and referenced against non-deuterated (1H)/deuterated (
13C) solvents. The shift values (δH and
δC) are always given in ppm with J values in Hz. The melting points were measured using a
Stanford Research Systems OptiMelt MPA 100. The high-resolution mass spectra were obtained
using a Finnigan MAT SSQ 710A spectrometer at 70 eV (HREIMS, positive and negative mode)
or an Agilent 6540 UHD (HRESIMS, positive and negative mode). Silica gel 60 M (40-63 µm,
Merck) was used for the flash column chromatography. The starting materials and reagents were
purchased from commercial suppliers and used without further purification. The solvents were
p.a. grade for the reaction mixtures and industrial grade for the flash column chromatography.
Analytical TLC was performed on silica gel coated alumina plates (MN TLC sheets ALUGRAM
® Xtra SIL G/UV254). The visualisation was performed using UV-light (254 and 366 nm). The
logP values were calculated using Marvinsketch 5.10.1.
Chapter 4 | 113
N-(5-Methylphenyl)cinnamamide (2)
E-3-Phenylpropenoyl chloride (1.00 g, 9.30 mmol) was stirred vigorously with p-toluidine (1.55
g, 9.30mmol) and K2CO3 (1.99 g, 14.4 mmol) in water (4.6 mL) and acetone (4.6 mL) at 0°C for
2 h. The mixture was then poured into ice-water (10 mL). The precipitate gave N-(5-
Methylphenyl)cinnamamide 2 as white powder. Yield: 2.21 g, 100%. 1H NMR (300MHz, CDCl3)
δ 7.75 (d, J = 15.6Hz, 1H, COCH), 7.50 − 7.54 (m, 4H, ArH), 7.37 − 7.39 (m, 3H, ArH), 7.16 (d,
J = 8.4Hz, 2H, ArH), 6.54 (d, J = 15.6Hz, 1H, ArCH), 2.33(s, 3H, CH3). The spectroscopic data
are in accordance with the literature values.[12]
6-Methylquinolin-2(1H)-one (3)
Compound 2 (1.00 g, 4.21 mmol) and AlCl3 (2.660 g, 19.95 mmol) were heated to 125°C in
chlorobenzene (10 mL) for 24 h. The mixture was cooled to 50°C and poured onto ice. The
mixture was extracted with EtOAc. Evaporation and recrystallization (EtOH) gave 6-
Methylquinolin-2(1H)-one 3 as pale yellow solid. Yield: 1.56 g, 37%. 1H NMR (300 MHz,
CDCl3) δ 12.18 (s, 1H, NH), 7.78 (d, J = 9.5 Hz, 1H, COCH), 7.36 (d, J = 4.6 Hz, 3H, ArH),
6.72 (d, J = 9.5 Hz, 1H, ArCH), 2.42 (s, 3H, CH3). The spectroscopic data are in accordance with
the literature values.[13]
2-Bromo-6-methylquinoline (4)
POBr3 (7.20 g, 37.0 mmol) was heated with 3 (2.00 g, 12.6 mmol) at 140°C for 3 h. The cooled
mixture was poured into ice-water. The precipitate was collected and dried. Chromatography on
silica gel (PE (50−70°C) : EtOAc = 25 : 1) gave 2-bromo-6-methylquinoline 4 as pale yellow
solid. Yield: 1.40 g, 50%. 1H NMR (300MHz, CDCl3) δ 7.90 − 7.95(m, 2H, ArH), 7.55 − 7.57(m,
2H, ArH), 7.48(d, J = 8.7Hz, 1H, ArH), 2.53(s, 3H, CH3).
1,2,3,4-Tetrahydroisoquinoline-6,7-diol hydrobromide (6)
The synthesis of compound 6 has already been described.[14]
To a mixture of HBr (24 mL, 48%
in H2O) and CH3COOH (96 mL) was added 6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline (4.21
Chapter 4 | 114
g, 21.8 mmol) and refluxed for 8 h. The solvent mixture was removed by distillation. The
product was used directly in the next step without further purification. Yield: 5.36 g, 100%. 1H
NMR (400 MHz, CD3OD) δ 2.95 (t, J = 6.3 Hz, 2H, CH2), 3.31 (quint, J = 1.7 Hz, 1H, NH),
3.42 (d, J = 6.4 Hz, 2H, CH2), 4.18 (s, 2H, CH2), 6.59 (s, 1H, ArH), 6.62 (s, 1H, ArH).
tert-Butyl 6,7-dihydroxy-3,4-dihydroisoquinoline-2(1H)-carboxylate (7)
Compound 7 was prepared according to known procedures [14]
from 6 (5.36 g, 21.8 mmol), di-
tertbutyldicarbonate (4.52 g, 20.7 mmol), and triethylamine (11.0 g, 109 mmol). Flash column
chromatography (PE (50−70°C) : EtOAc = 1:1) of the sticky brown crude product yielded a
slightly yellow solid. Yield: 4.51 g, 78 %. 1H NMR (400 MHz, CDCl3) δ 1.48 (s, 9H), 2.66 (t, J
= 5.6 Hz, 2H), 3.58 (s, 2H), 4.41 (s, 2H), 6.55 (s, 1H), 6.58 (s, 1H).
tert-Butyl 6,7-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-3,4-dihydroisoquinoline- 2(1H)-
carboxylate (8)
Compound 7 (4.51 g, 17.0 mmol), potassium hydroxide (1.74 g, 31.0 mmol), and 2-(2-(2-
Methoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (9.87 g, 31.0 mmol) were dissolved in
tetrahydrofurane and refluxed overnight. The solvent was evaporated and the residue was taken
up in ethyl acetate, washed with water and brine, and concentrated. Flash column
chromatography (EtOAc) of the crude product yielded 8 as yellow oil. Yield: 5.30 g, 73%. 1H
NMR (400 MHz, CDCl3) δ 1.41 (s, 9 H, tBu), 2.65 (t, J = 5.3 Hz, 2H), 3.30 (s, 6 H, OCH3), 3.46
− 3.49 (m, 4 H, PEG), 3.51-3.56 (m, 2H), 3.56 − 3.61 (m, 8H, PEG), 3.65 − 3.68 (m, 4H, PEG),
3.77 (t, J = 5.1 Hz, 4H, PEG), 4.06 (t, J = 5.04 Hz, 4H, PEG), 4.39 (s, 2H), 6.57 (s, 1H), 6.60 (s,
1H). 13
C NMR (101 MHz, CDCl3) δ 28.4, 28.5, 40.6, 41.9, 44.9, 45.5, 59.0, 69.0, 69.1, 69.6,
70.5, 70.7, 70.8, 71.9, 79.7, 112.7, 115.1, 126.2, 127.6, 147.5, 147.5, 154.9.
6,7-Bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-1,2,3,4-tetrahydroisoquinoline 2,2,2-
trifluoroacetate (9)
Chapter 4 | 115
Compound 8 (5.02 g, 9.0 mmol) was dissolved in DCM and trifluoroacetic acid (10.26 g, 90
mmol) and stirred overnight at room temperature. The solvent was evaporated to yield 9 as a
gray solid, which was used without further purification. Yield: 5.14 g, 100%. Mp. 47°C (45.4 –
48.0°C).1H NMR (400 MHz, CD3OD) δ 3.00 (t, J =6.2 Hz, 2H), 3.35 (s, 6H), 3.49 (t, J = 6.4 Hz,
2H), 3.52 − 3.56 (m, 4H, PEG), 3.63 − 3.68 (m, 8 H, PEG), 3.71 − 3.74 (m, 4H, PEG), 3.82-3.85
(m, 4H, PEG), 4.07 − 4.13 (m, 4H, PEG), 4.25 (s, 2H), 6.68 (s, 1H), 6.73 (s, 1H). 13
C NMR (101
MHz, CD3OD) δ 25.6, 43.1, 45.6, 59.2, 70.2, 70.3, 70.9, 71.4, 71.7, 71.8, 72.9, 113.9, 115.8,
122.0, 125.7, 149.3, 150.0. HRMS (EI-MS) [M+H]+ calcd. for C23H40NO8 458.2748, found
458.2757.
6-Methoxy-1,2,3,4-tetrahydroisoquinolin-7-ol hydrochloride (14)
Compound 14 was prepared according to literature-known procedures.[8a]
The catalyst was
removed by centrifugation and the crude product was recrystallized from ethanol. Yield: 4.38 g,
44%. 1H NMR (400 MHz, MeOD) δ 3.02 (t, J = 6.2 Hz, 2H), 3.45 (t, J = 6.3 Hz, 2H), 3.84 (s,
3H), 4.20 (s, 2H), 6.61 (s, 1H), 6.77 (s, 1H).
tert-Butyl 7-hydroxy-6-methoxy-3,4-dihydroisoquinoline-2(1H)-carboxylate (15)
Compound 15 was prepared from 14 (4.38 g, 20.3 mmol) according to literature-known
procedures.[8b]
Flash column chromatography (PE (50−70°C) : EtOAc = 3:2) of the sticky brown
crude product yielded 15 as colorless oil. Yield: 3.12 g, 55%. 1H NMR (400 MHz, CDCl3) δ 1.48
(s, 9H), 2.73 (t, J = 5.4 Hz, 2H), 3.61 (s, 2H), 3.85 (s, 3H), 4.45 (s, 2H), 6.59 (s, 1H), 6.64 (s,
1H).
tert-Butyl 6-methoxy-7-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-3,4- dihydroisoquinoline-2(1H)-
carboxylate (16)
Compound 16 was prepared from 15 (0.20 g, 0.72 mmol) according to literature-known
procedures.[8b]
The crude product was purified with flash column chromatography (PE
(50−70°C) : EtOAc = 3:2) to obtain a brownish oil. Yield: 0.26 g, 86%. 1H NMR (400 MHz,
CDCl3) δ 1.48 (s, 9H), 2.73 (t, J = 5.2 Hz, 2H), 3.37 (s, 3H), 3.53-3.56 (m, 2H), 3.59-3.69 (m,
Chapter 4 | 116
6H), 3.71-3.75 (m, 2H), 3.82 (s, 3H), 3.86 (t, J = 5.2 Hz, 2H), 4.14 (t, J = 5.2 Hz, 2H), 4.46 (s,
2H), 6.60 (s, 1H), 6.64 (s, 1H).
tert-Butyl 6-methoxy-7-(2-morpholinoethoxy)-3,4-dihydroisoquinoline-2(1H)-carboxylate (17)
The method for preparation of Compound 17 was described in the literature.[8b]
1H NMR (300
MHz, CDCl3) δ 6.61 (s, 2H, ArH), 4.46 (s, 2H, CONCH2Ar), 4.11 (t, J = 6.0 Hz, 2H, OCH2Ar),
3.82 (s, 3H, OCH3), 3.77 – 3.70 (m, 4H, OCH2, OCH2), 3.64 − 3.60( m, 2H, NCH2), 2.83 (t, J =
6.0 Hz, 2H, CH2Ar), 2.74 (t, J = 5.7 Hz, 2H, NCH2), 2.62 – 2.54 (m, 4H, NCH2, NCH2), 1.48 (s,
9H, tBu).
13C NMR (151 MHz, CDCl3) δ 154.9, 148.1, 146.7, 127.2, 125.5, 112.0, 111.6, 79.7,
66.8(2C), 60.4, 57.5, 56.0, 54.0(2C), 45.6, 44.9, 29.7, 28.5(3C). HRMS (EI-MS) [M+H]+ calcd
for C21H32N2O5 393.2384, found 393.2384.
tert-Butyl6-methoxy-7-(3-morpholinopropoxy)-3,4-dihydroisoquinoline-2(1H)-carboxylate (18)
The method for preparation of Compound 18 was described in the literature.[8b] 1
H NMR (300
MHz, CDCl3) δ 6.54 (d, J = 3.7 Hz, 2H), 4.39 (s, 2H), 3.74 (s, 3H), 3.64-3.61 (m, 4H), 3.55-3.51
(m,, 2H), 2.65 (t, J = 5.6 Hz, 2H), 2.36 – 2.35 (m, 6H), 1.98 – 1.86 (m, 2H), 1.40 (s, 9H). 13
C
NMR (75 MHz, CDCl3) δ 154.8, 148.0, 147.0, 126.7, 125.2, 112.0, 111.1, 79.6, 67.4, 66.9(2C),
56.0, 55.7, 55.4, 53.7(2C), 43.0, 28.5(3C), 26.3. HRMS (EI-MS) (m/z) [M+H]+ calcd for
C22H34N2O5 407.2540, found 407.2551.
tert-Butyl 6-methoxy-7-(2-(piperidin-1-yl)ethoxy)-3,4-dihydroisoquinoline-2(1H)-carboxylate
(19)
The method for preparation of Compound 19 was described in the literature.[8b] 1
H NMR (300
MHz, CDCl3) δ 6.62 (d, J = 3.4 Hz, 2H, ArH), 4.47 (s, 2H, CONCH2Ar), 4.17 − 4.14 (m, 2H,
OCH2), 3.83 (s, 3H, OCH3), 3.61 (t, J = 5.0Hz, 2H, CONCH2), 2.85 (m, 2H, CH2Ar), 2.74 (t, J =
5.7Hz, 2H, NCH2), 2.57 (s, br, 4H, NCH2, NCH2), 1.74 – 1.55 (m, 6H, CH2CH2CH2), 1.49 (s, 9H,
tBu).
13C NMR (151 MHz, CDCl3) δ 154.9, 148.0, 146.7, 127.3, 125.4, 112.0, 111.5, 79.8, 66.4,
57.5, 56.0(2C), 54.8(2C), 49.9, 45.5, 28.8, 28.5(3C), 25.4, 23.8. HRMS (EI-MS) [M+H]+ calcd
for C22H34N2O4 391.2591, found 391.2597.
Chapter 4 | 117
tert-Butyl 6-methoxy-7-(3-(piperidin-1-yl)propoxy)-3,4-dihydroisoquinoline-2(1H)-carboxylate
(20)
The method for preparation of Compound 20 was described in the literature.[8b] 1
H NMR (600
MHz, CDCl3) δ 6.61 (d, J = 6.5 Hz, 2H, ArH), 4.47 (s, 2H, CONCH2Ar), 4.04 (t, J = 6.3 Hz, 2H,
OCH2), 3.82 (s, 3H, OCH3), 3.61 (s, br, 2H, NHCH2), 2.73 (s, 2H, ArCH2), 2.52 – 2.05 (m, 6H,
NCH2, NCH2, NCH2), 2.06(m, 2H, CH2), 1.71 – 1.62 (m, 6H, CH2CH2CH2), 1.48 (s, 9H). 13
C
NMR (151 MHz, CDCl3) δ 154.9, 148.0, 147.0, 127.0, 125.8, 125.3, 112.0, 111.2, 79.7, 67.7,
60.4, 56.1, 55.8, 54.4(2C), 44.7, 29.7, 28.5(3C), 26.2, 25.4, 24.0. HRMS (EI-MS) [M+H]+ calcd
for C23H36N2O4 405.2748, found 405.2754.
tert-Butyl-6-methoxy-7-(2-(pyrrolidin-1-yl)ethoxy)-3,4-dihydroisoquinoline-2(1H)-carboxylate
(21)
The method for preparation of Compound 21 was described in the literature.[8b] 1
H NMR (300
MHz, CDCl3) δ 6.55 (d, J = 3.3 Hz, 2H, ArH), 4.40 (s, 2H, CONCH2Ar), 4.07 (t, J = 6.2Hz, 2H,
OCH2), 3.75 (s, 3H, OCH3), 3.59 – 3.55 (m, 2H, CONCH2), 2.92 (t, J = 6.2 Hz, 2H, CH2Ar),
2.69 − 2.64 (m, 6H, NCH2, NCH2, NCH2), 1.74 − 1.79 (m, 4H, CH2CH2), 1.41 (s, 9H, tBu). 13
C
NMR (75 MHz, CDCl3) δ 153.9, 146.5, 145.7, 126.2, 124.4, 110.9, 110.3, 78.8, 66.7, 61.5, 55.0,
53.7, 53.5(3C), 29.1, 27.5(3C), 22.4(2C). HRMS (EI-MS) [M+H]+ calcd for C21H32N2O4
377.2435, found 377.2432.
6-Methoxy-7-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-1,2,3,4-tetrahydroisoquinoline 2,2,2-
trifluoroacetate (22)
Compound 22 has already been described.[8b]
The boc-protected compound 16 (3.83 g, 9.0 mmol)
was dissolved in DCM, then trifluoroacetic acid (2.61 g, 36.0 mmol) was added. The solution
was stirred overnight and the solvent was removed by evaporation. The sticky brown oil 22 was
used without purification. Yield: 3.95 g, 100%. 1H NMR (400 MHz, MeOD) δ 3.03 (t, J = 6.3 Hz,
2 H), 3.35 (s, 3 H), 3.46 (t, J = 6.4 Hz, 2 H), 3.52 − 3.55 (m, 2 H), 3.62 − 3.66 (m, 4 H), 3.69 −
3.72 (m, 2 H), 3.81 (s, 3 H), 3.82 − 3.84 (m, 2 H), 4.09 − 4.12 (m, 2 H), 4.25 (s, 2 H), 6.78 (s, 1
H), 6.80 (s, 1 H).
Chapter 4 | 118
4-(2-((6-Methoxy-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)ethyl)morpholine (23)
Compound 23 has already been described.[8b]
1H NMR (300 MHz, MeOD) δ 6.65 (d, J = 9.3 Hz,
2H, ArH), 4.07 (t, J = 5.6 Hz, 2H, OCH2), 3.90 – 3.80 (s, 2H, HNCH2Ar), 3.77 (s, 3H, OCH3),
3.73 – 3.64 (m, 4H, OCH2, OCH2), 3.02 (t, J = 6.0 Hz, 2H, HNCH2), 2.82 – 2.69 (m, 4H, NCH2,
CH2Ar), 2.63 – 2.54 (m, 4H, NCH2, NCH2). 13
C NMR (75 MHz, MeOD) δ 149.8, 148.0, 128.6,
128.2, 113.9, 113.4, 68.3, 67.7(2C), 58.8, 56.5, 55.3(2C), 48.1, 44.5, 29.0. HRMS (EI-MS)
[M+H]+ calcd for C16H24N2O3 293.1859, found 293.1860.
4-(3-((6-Methoxy-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)propyl)morpholine (24)
Compound 24 has already been described.[8b]
1H NMR (300 MHz, MeOD) δ 6.63 (d, J = 12.7 Hz,
2H), 4.43 (s, NH, 1H), 3.97 (t, J = 6.2 Hz, 2H), 3.85 (s, 2H), 3.76 (s, 3H), 3.72 – 3.63 (m, 4H), ,
3.03 (t, J = 6.0 Hz, 2H), 2.73 (t, J = 5.6 Hz, 2H), 2.56 – 2.40 (m, 6H), 2.01 – 1.86 (m, 2H). 13
C
NMR (75 MHz, MeOD) δ 149.7, 148.3, 127.9, 127.9, 113.9, 112.8, 68.5, 67.7, 67.7, 56.9, 56.6,
54.9, 48.0, 44.4, 28.9, 28.8, 27.4. HRMS (EI-MS) [M+H]+ calcd for C17H26N2O3 307.2016, found
307.2020.
6-Methoxy-7-(2-(piperidin-1-yl)ethoxy)-1,2,3,4-tetrahydroisoquinoline (25)
Compound 25 has already been described.[8b] 1
H NMR (300 MHz, MeOD) δ 6.89 (d, J = 5.5 Hz,
2H, ArH), 4.31 (t, J = 4.8Hz, 2H, OCH2), 4.27 (s, 2H, NHCH2Ar), 3.87 (s, 3H, OCH3), 3.70 (d, J
= 12.4 Hz, 2H, NHCH2), 3.54 (t, J = 5.1Hz, 2H, CH2Ar), 3.47 (t, J = 6.4 Hz, 2H, NCH2), 3.10 –
3.02 (m, 4H, NCH2, NCH2), 2.01 − 1.80 (m, 6H, CH2CH2CH2). 13
C NMR (75 MHz, MeOD) δ
151.0, 147.6, 127.2, 121.4, 114.5, 113.4, 65.2, 57.2, 56.5, 55.0(2C), 45.3, 42.8, 25.8, 24.2(2C),
22.6. HRMS (EI-MS) [M+H]+ calcd for C17H26N2O2 291.2072, found 291.2068.
6-Methoxy-7-(3-(piperidin-1-yl)propoxy)-1,2,3,4-tetrahydroisoquinoline (26)
Compound 26 has already been described.[8b]
1H NMR (600 MHz, MeOD) δ 6.85 (s, 1H, ArH),
6.80 (s, 1H, ArH), 4.25 (s, 2H, NHCH2Ar), 4.10 (t, J = 5.6 Hz, 2H, OCH2), 3.84 (s, 3H, OCH3),
3.64 (d, J = 12.1 Hz, 2H, NHCH2), 3.46 (t, J = 6.4 Hz, 2H, ArCH2), 3.35 – 3.32 (m, 2H, NCH2),
3.04 (t, J = 6.3 Hz, 2H, NCH2), 2.96 (td, J 1= 12.5, J2 = 2.6 Hz, 2H, NCH2), 2.29 – 2.18 (m, 2H,
CH2), 2.03 – 1.94 (m, 2H, CH2), 1.84 – 1.73 (m, 2H, CH2), 1.54 (qt, J1 = 13.2, J2=3.9 Hz, 2H,
Chapter 4 | 119
CH2). 13
C NMR (151 MHz, MeOD) δ 150.7, 148.4, 125.9, 121.1, 113.1, 112.6, 79.5, 68.1, 56.7,
56.4, 54.6, 45.4, 42.9, 25.7, 25.1, 24.4(2C), 22.7. HRMS (EI-MS) [M+H]+ calcd for C18H28N2O2
305.2224, found 305.2224.
6-Methoxy-7-(2-(pyrrolidin-1-yl)ethoxy)-1,2,3,4-tetrahydroisoquinoline (27)
Compound 27 has already been described.[8b]
1H NMR (300 MHz, MeOD) δ 6.68 (s, 1H), 6.64 (s,
1H), 4.08 (t, J = 5.8 Hz, 2H), 3.87 (s, 2H), 3.78 (s, 3H), 3.05 (t, J = 6.0 Hz, 2H), 2.91 (t, J = 5.8
Hz, 2H), 2.72 (m, 6H), 1.88 – 1.77 (m, 4H). 13
C NMR (75 MHz, MeOD) δ 149.8, 148.0, 128.4,
127.9, 113.9, 113.2, 69.2, 56.5, 56.0, 55.7(2C), 48.0, 44.4, 28.8, 24.3(2C). HRMS (EI-MS)
[M+H]+ calcd for C16H24N2O2 277.1911, found 277.1915.
Methyl 2-amino-4-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)benzoate (29)[15]
A mixture of Pd(dppf)Cl2 (0.270 g, 0.331 mmol), KOAc (1.95 g, 19.870 mmol), bis(neopentyl
glycolato)diboron (1.80 g, 7.968 mmol), and methyl 2-amino-4-bromobenzoate 37 (1.50 g, 6.551
mmol) was added to a flask under anhydrous conditions. After addition of anhydrous DMSO, the
mixture was stirred at 80ºC for several hours and the reaction progress was checked by TLC. The
reaction solution was cooled to room temperature and poured into ice-water. The mixture was
extracted with ethyl acetate and the combined organic layers were washed with saturated brine,
dried over Na2SO4, and concentrated in vacuo. The residue was purified by flash column
chromatography to give the corresponding aryl boronate as white solid. Yield: 1.55 g, 90%. 1H
NMR (300 MHz, CDCl3) δ 7.82 (d, J = 8.0 Hz, 1H, ArH), 7.12 (s, 1H, ArH), 7.04 (d, J = 8.0 Hz,
1H, ArH), 5.46 (s, br, 2H, NH2), 3.86 (s, 3H, OCH3), 3.76 (s, 4H, CH2, CH2), 1.02 (s, 6H, CH3,
CH3). 13
C NMR (75 MHz, CDCl3) δ 167.7, 148.5, 129.0(2C), 121.5, 120.2, 111.3, 71.3(2C), 50.5,
30.8, 20.8, 20.8. HRMS (EI-MS) [M•+] calcd for C13H18BNO4 262.1365, found 262.1361.
Methyl 2-amino-4-(6-methylquinolin-2-yl)benzoate (30)[16]
2-Bromo-6-methylquinoline 4 (1.38 g, 6.21 mmol), methyl 2-amino-4-(5, 5-dimethyl-1, 3, 2-
dioxaborinan-2-yl) benzoate 29 (1.50 g, 5.70 mmol) and [Pd(PPh3)4] (0.72 g, 0.63 mmol) were
placed into a Schlenk flask under a stream of nitrogen at room temperature. The mixture of
Chapter 4 | 120
solids was stirred and degassed three times before it was dissolved in anhydrous and degassed
THF (10 mL), aqueous K3PO4 (2 mol/L) was added and the reaction mixture was heated to 80°C
overnight. The resulting dark-brown reaction mixture was cooled to room temperature and
diluted with water (10 mL). After extracted with CH2Cl2, washed with brine and concentrated in
vacuo, the residue was purified by flash column chromatography over silica gel (PE (50−70°C) :
EtOAc = 1:1) to give methyl 2-amino-4-(6-methylnaphthalen-2-yl)benzoate 30 as white solid.
Yield: 1.09 g, 60%. 1H NMR (300 MHz, CDCl3) δ 8.15 (d, J = 8.6 Hz, 1H, ArH), 8.10 (d, J = 8.4
Hz, 1H, ArH), 7.99 (d, J = 8.4 Hz, 1H, ArH), 7.82 (d, J = 8.6 Hz, 1H, ArH), 7.60 (s, 1H, ArH),
7.57 (d, J = 1.9 Hz, 1H, ArH), 7.55(d, J = 1.6Hz, 1H, ArH), 7.35 (dd, J1 = 8.4Hz, J2 = 1.7 Hz, 1H,
ArH), 5.87 (s, br, 2H, NH2), 3.91 (s, 3H, OCH3), 2.56 (s, 3H, ArCH3). 13
C NMR (75 MHz,
CDCl3) δ 167.4, 154.4, 149.7, 145.6, 143.7, 135.6, 135.2, 131.1, 130.8, 128.3, 126.5, 125.3,
118.1, 114.5, 114.3, 110.1, 50.6, 20.6. HRMS (EI-MS) [M+H]+ calcd for C18H16N2O2 293.1285,
found 293.1290.
Methyl 4-(6-methylquinolin-2-yl)-2-(quinoline-2-carbonylamino)benzoate (31)
Quinoline-2-carboxylic acid (1.0 equiv) was suspended in SOCl2 (10−15 mL) and heated to
reflux for 2 h. Excess SOCl2 was removed under reduced pressure and the resulting quinoline-2-
carbonyl chloride was obtained as yellow solid. Methyl 2-amino- 4-(6-methylnaphthalen-2-
yl)benzoate 30 (1.0 equiv) and NEt3 (1.2 equiv) were dissolved in CH2Cl2 and the freshly
prepared quinoline-2-carbonyl chloride derived was added in small portions and stirred at room
temperature for 30 min. Then, the solution was refluxed at 40°C overnight, washed with 1N HCl
and saturated aqueous solution of Na2CO3 (3×), dried over anhydrous Na2SO4 and concentrated
to give the crude product which was purified by flash chromatography (PE (50−70°C) : EtOAc =
4:1) on silica gel to give 31 as white solid. 1H NMR (600 MHz, CDCl3) δ 13.38 (s, 1H, NH),
9.79 (d, J = 1.6 Hz, 1H, ArH), 8.43 (d, J = 8.4 Hz, 1H, ArH), 8.40 (s, 1H, ArH), 8.38 (d, J = 3.9
Hz, 1H, ArH), 8.36 (s, 1H, ArH), 8.30 (d, J = 8.3 Hz, 1H, ArH), 8.23 (d, J = 8.5 Hz, 1H, ArH),
8.14 (d, J = 7.5 Hz, 1H, ArH), 8.05 (d, J = 8.6 Hz, 1H, ArH), 7.93 (d, J = 8.1 Hz, 1H, ArH), 7.86
– 7.82 (m, 1H, ArH), 7.69 – 7.65 (m, 1H, ArH), 7.64 (s, 1H, ArH), 7.62 – 7.59 (m, 1H, ArH),
4.11 (s, 3H, OCH3), 2.57 (s, 3H, ArCH3). 13
C NMR (151 MHz, CDCl3) δ 167.9, 163.8, 154.9,
150.1, 146.7, 141.2, 137.7, 137.1(2C), 136.9(2C), 132.5(2C), 131.9, 130.3, 130.2, 129.4, 128.3,
127.7, 126.4, 122.2(2C), 119.5, 119.5, 118.9, 116.9, 52.5, 21.7. HRMS (EI-MS) [M+H]+ calcd
Chapter 4 | 121
for C28H21N3O3 448.1656, found 448.1658.
Methyl 4-(6-(bromomethyl)quinolin-2-yl)-2-(quinoline-2-carbonylamino)benzoate (32)[17]
A solution of methyl 4-(6-methylnaphthalen-2-yl)-2-(quinoline-2-carboxamido)benzoate 31 (1g,
2.23 mmol), 0.48 g (0.268 mmol) of NBS, and 0.03 g (0.12 mmol) of dibenzoyl peroxide in 50
mL of carbon tetrachloride was refluxed for 6 h. After the reaction mixture was filtered, it was
washed with aqueous NaHCO3 and brine. The solvent was evaporated to obtain compound 32
which was sufficiently pure to be used without further purification. Yield: 0.61 g, 52%, white
solid. 1H NMR (600 MHz, CDCl3) δ 13.36 (s, 1H, -CONH), 9.79 (d, J = 1.5 Hz, 1H, ArH), 8.41
(d, J = 8.4 Hz, 1H, ArH), 8.38 – 8.36 (m, 1H, ArH), 8.35 (d, J = 8.5 Hz, 1H, ArH), 8.27 (d, J =
8.3 Hz, 1H, ArH), 8.23 (d, J = 3.4 Hz, 1H, ArH), 8.22 (d, J = 3.5 Hz, 1H, ArH), 8.11 – 8.08 (m,
1H, ArH), 8.07 (d, J = 8.6 Hz, 1H, ArH), 7.91 (d, J = 7.8 Hz, 1H, ArH), 7.85 – 7.81 (m, 2H,
ArH), 7.77 (dd, J = 8.7, 1.9 Hz, 1H, ArH), 7.66 (t, J = 7.3 Hz, 1H, ArH), 4.68 (s, 2H, CH2Br),
4.10 (s, 3H, OCH3). 13
C NMR (151 MHz, CDCl3) δ 167.8, 163.8, 156.4, 150.0, 147.7, 146.6,
144.5, 141.2, 137.7, 137.1, 136.2, 131.8, 130.9, 130.6, 130.3, 130.2, 129.4, 128.3, 127.7, 127.4,
127.3, 126.6, 122.1, 119.9, 119.5, 118.9, 117.0, 52.5. HRMS (EI-MS) [M+H] +
calcd for
C28H20BrN3O3 526.0761, found 526.0764.
General procedure for the preparation of compounds 33a-h
Tetrahydroisoquinoline derivatives (1.0 equiv.), methyl 4-(6-(bromomethyl) naphthalen-2-yl)-2-
(quinoline-2-carboxamido) benzoate 32 (1.0 equiv.) and diisopropylethylamine (2.0 equiv.) were
dissolved in CH3CN and the mixture was refluxed overnight. Flash column chromatography
(CHCl3:CH3OH = 20:1) gave the corresponding products.
Methyl4-(6-((6,7-dimethoxy-3, 4-dihydroisoquinolin-2(1H)-yl)methyl)quinolin -2-yl)-2-
(quinoline-2-carbonylamino)benzoate (33a)
Compound 33a according to the general procedure. The crude product was purified with flash
column chromatography (EtOAc→CHCl3 : MeOH = 20:1) to obtain 33a as pale yellow solid.
Mp. 158°C (decomposition), 1H NMR (600 MHz, CDCl3) δ 13.4(s, 1H, -CONH), 9.82(d, J = 1.7
Chapter 4 | 122
Hz, 1H, ArH), 8.44(d, J = 8.4Hz, 1H, ArH), 8. 39(d, J = 8.5Hz, 1H, ArH), 8.37(d, J = 8.5Hz,1H,
ArH), 8.29 (d, J = 8.3 Hz, 1H, ArH), 8.27 (d, J = 8.6 Hz, 1H, ArH), 8.20 (d, J = 8.6 Hz, 1H,
ArH), 8.11 (dd, J1 = 8.3, J2 = 1.8 Hz, 1H, ArH), 8.08 (d, J = 8.6 Hz, 1H, ArH), 7.93 (d, J = 8.0
Hz, 1H, ArH), 7.84 (ddd, J1 = 8.3, J2 = 6.9, J3 = 1.3 Hz, 2H, ArH), 7.78 (d, J = 8.6Hz, 1H, ArH),
7.68 (ddd, J1 = 8.0, J2 = 6.9, J3 = 1.1 Hz, 1H, ArH), 6.63(s,1H, ArH), 6.49(s, 1H, ArH), 4.11
(s,3H, OCH3), 3.86-3.85(m, 2H, -NCH2Ar), 3.9 (s, 3H, ArOCH3), 3.8 (s, 3H, ArOCH3), 3.70 (s,
br, 2H, ArCH2N-), 2.89-2.86 (m,4H, -NCH2CH2Ar). 13
C NMR (151 MHz, CDCl3) δ 167.9, 163.8,
160.1, 154.7, 150.1, 147.9, 147.4, 146.7, 145.1, 141.2, 137.7(2C), 136.8, 131.8, 131.3, 130.3,
130.2, 129.4, 128.3, 127.7 (2C), 127.4, 126.4, 122.0, 119.6, 119.5 (2C), 118.9 (2C), 116.8, 111.4,
109.5, 55.9, 55.9, 53.4, 52.5 (2C), 48.5, 28.2. HRMS (EI-MS) [M+H]+ calcd for C46H47N5O5
639.2602, found 639.2604.
Methyl4-(6-((6-methoxy-7-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-3,4-dihydroisoquinolin-
2(1H)-yl)methyl)quinolin-2-yl)-2-(quinoline-2-carbonylamino)benzoate (33b)
Compound 33b was prepared according to the general procedure. The crude product was purified
with flash column chromatography (EtOAc→CHCl3 : MeOH = 20:1) to obtain 33b as pale
yellow solid. Mp.102°C (decomposition), 1H NMR (400 MHz, CDCl3) δ 13.37 (s, 1H, -CONH),
9.81 (d, J = 1.7 Hz, 1H, ArH), 8.43 (d, J = 8.5 Hz, 1H, ArH), 8.38 (d, J = 8.1 Hz, 1H, ArH), 8.36
(d, J = 7.8 Hz, 1H, ArH), 8.28 (d, J = 8.4 Hz, 1H, ArH), 8.25 (d, J = 8.6 Hz, 1H, ArH), 8.19 (d, J
= 8.6 Hz, 1H, ArH), 8.11 (dd, J = 8.4, 1.8 Hz, 1H, ArH), 8.07 (d, J = 8.6 Hz, 1H, ArH), 7.92 (dd,
J1 = 8.2, J2=0.9 Hz, 1H, ArH), 7.84 (m, 3H, ArH), 7.67 (m, 1H, ArH), 6.62 (s, 1H, ArH), 6.55 (s,
1H, ArH), 4.12 − 4.09(m, 5H, ArOCH3, ArOCH2), 3.89(s, 2H, -NCH2Ar), 3.85-3.82(m, 5H,
PEG), 3.72 − 3.69(m, 2H, -NCH2Ar), 3.66 − 3.61(m, 5H, PEG), 3.53 − 3.50 (m, 2H, PEG),
3.35(s, 3H, OCH3), 2.87 − 2.83(m, 4H, -NCH2CH2). 13
C NMR (101 MHz, CDCl3) δ 167.9, 163.8,
155.7, 150.1, 148.3, 147.9, 146.6, 146.6, 145.1, 141.2, 137.7, 136.8, 131.8, 131.3, 130.5, 130.3,
130.2, 130.0, 129.4, 128.3, 127.7, 127.2, 127.1, 126.8, 122.0, 119.5(2C), 118.9, 116.8 , 112.4,
112.1, 71.9, 70.8, 70.6, 70.5, 69.6, 68.7, 62.4, 59.0, 56.0, 55.6, 53.4, 52.5, 50.8, 28.6. HRMS (EI-
MS) [M+H] +
calcd for C45H46N4O8 771.3394, found 771.3388.
Chapter 4 | 123
Methyl4-(6-((6,7-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-3,4-dihydroisoquinolin-2(1H)-yl)-
methyl)quinolin-2-yl)-2-(quinoline-2-carbonylamino)benzoate (33c)
Compound 33c was prepared according to the general procedure. The crude product was purified
with flash column chromatography (EtOAc→CHCl3 : MeOH = 20:1) to obtain 26 as pale yellow
solid. Mp.108°C（decomposition）,1H NMR (300 MHz, CDCl3) δ 13.59 (s, 1H, -CONH), 9.81
(d, J = 1.7 Hz, 1H, ArH), 8.43(d, J = 8.5Hz, 1H, ArH), 8.38 (d, J = 8.4 Hz, 1H, ArH), 8.36 (d, J
= 8.4Hz, 1H, ArH), 8.29 − 8.23 (dd, J1 = 8.6 Hz, J2 = 9.4 Hz, 2H, ArH), 8.19 (d, J = 8.6, 1H,
ArH), 8.12 − 8.08 (dd, J1 = 8.4, J2=1.8 Hz, 1H, ArH)， 8.07 (d, J = 8.6Hz, 1H, ArH)， 7.93 (d,
J = 8.1, 1H, ArH), 7.86 − 7.83 (dd, J1 = 6.8Hz, J2 = 1.4 Hz, 1H, ArH) , 7.85 − 7.80 (dd, J1 = 8.6,
J2 = 1.7Hz, 2H, ArH), 7.69 − 7.61(m, 1H, ArH), 6.67(s, 1H, ArH), 6.54 (s, 1H, ArH), 4.14 − 4.05
(m, 7H, ArOCH3, ArOCH2), 3.88 (s, 2H, -NCH2), 3.85 − 3.80 (dd, J1 = 8.7Hz, J2 = 5.3Hz, 2H, -
NCH2Ar), 3.82 − 3.79(d, J = 8.7 Hz, 2H, PEG), 3.75-3.60(m, 14H, PEG), 3.66 − 3.50 (m, 4H,
PEG), 3.37 (s, 3H, OCH3)，3.36 (s, 3H, OCH3), 2.85(s, br, 4H, -NCH2CH2). 13
C NMR (75 MHz,
CDCl3) δ 166.9, 162.8, 154.7, 149.1, 146.8, 146.5, 146.2, 145.6, 144.1, 140.2, 136.7, 135.8,
130.8, 130.3, 129.3, 129.2, 129.0, 128.4, 127.3, 126.7, 126.4, 126.0(2C), 121.0, 118.5, 118.4,
117.9, 115.7, 114.1, 112.2, 70.9, 70.9, 69.8, 69.8, 69.7, 69.7, 69.5, 69.5, 68.7, 68.7, 68.0, 61.4,
58.0, 58.0, 54.6, 51.6, 49.8, 28.7, 28.5. HRMS (EI-MS) [M+H]+ calcd for C51H58N4O11 903.4175,
found 903.4173.
Methyl4-(6-((6-methoxy-7-(2-morpholinoethoxy)-3,4-dihydroisoquinolin-2(1H)-yl) methyl)
quinolin-2-yl)-2-(quinoline-2-carbonylamino)benzoate (33d)
Compound 33d was prepared according to the general procedure. The crude product was purified
with flash column chromatography (EtOAc→CHCl3 : MeOH = 20:1) to obtain 33d as pale
yellow solid. Mp. 158°C (decomposition ), 1H NMR (600 MHz, CDCl3) δ 13.38 (s, 1H, -CONH),
9.81 (d, J = 1.7 Hz, 1H, ArH), 8.43 (d, J = 8.4 Hz, 1H, ArH), 8.39 (d, J = 8.5 Hz, 1H, ArH), 8.37
(d, J = 8.6 Hz, 1H, ArH), 8.29 (d, J = 8.3 Hz, 1H, ArH), 8.25 (d, J = 8.5 Hz, 1H, ArH), 8.20 (d, J
= 8.6 Hz, 1H, ArH), 8.10 (dd, J = 8.3, 1.7 Hz, 1H, ArH), 8.07 (d, J = 8.6 Hz, 1H, ArH), 7.92 (d,
J = 8.0 Hz, 1H, ArH), 7.87 (s, 1H, ArH), 7.84 (ddd, J1 = 8.8Hz, J2 = 5.8Hz, J3 = 4.6Hz, 2H, ArH),
7.68 − 7.67 (m, 1H, ArH), 6.63 (s, 1H, ArH), 6.52 (s, 1H, ArH). 4.11 (s, 3H, OCH3), 4.09 (t, J =
6.0 Hz, 2H, OCH2), 3.91 (s, 2H, -NCH2Ar), 3.82 (s, 3H, ArOCH3), 3.75 – 3.71 (m, 4H, OCH2,
Chapter 4 | 124
OCH2), 3.63 (s, 2H, -NCH2Ar), 2.88 (d, J = 5.0 Hz, 2H, CH2Ar), 2.87 – 2.80 (m, 4H, -NCH2, -
NCH2), 2.60 (s, br, 4H, -NCH2, -NCH2). 13
C NMR (151 MHz, CDCl3) δ 167.9, 163.8, 155.8,
150.1, 148.3, 147.9, 146.6, 146.4, 145.1, 141.2, 137.7, 136.8, 131.8, 131.3, 130.3 , 130.2, 130.1,
129.4, 128.3, 127.7, 127.4, 127.2, 126.8, 126.3, 122.0, 119.5, 119.5, 118.9, 116.8, 112.1, 112.0,
66.7, 62.3, 57.4, 56.0, 55.5, 53.9, 52.5, 50.8, 29.7, 28.8, 28.5, 19.6, 19.2. HRMS (EI-MS)
[M+H]+ calcd for C44H43N5O6 738.3286, found 738.3293.
Methyl4-(6-((6-methoxy-7-(3-morpholinopropoxy)-3,4-dihydroisoquinolin-2(1H)-yl) methyl)-
quinolin-2-yl)-2-(quinoline-2-carbonylamino)benzoate (33e)
Compound 33e was prepared according to the general procedure. The crude product was purified
with flash column chromatography (EtOAc→CHCl3 : MeOH = 20:1) to obtain 33e as pale
yellow solid. Mp. 155°C (decomposition), 1H NMR (300 MHz, CDCl3) δ 13.38 (s, 1H, -CONH),
9.80 (s, 1H, ArH), 8.42 (d, J = 8.5 Hz, 1H, ArH), 8.37 (dd, J1 = 9.4, J2 = 5.4 Hz, 2H, ArH), 8.27
(dd, J1 = 14.4, J2 = 6.0 Hz, 2H, ArH), 8.19 (d, J = 8.8 Hz, 1H, ArH), 8.12 – 8.03 (m, 2H, ArH),
7.91 (d, J = 8.1 Hz, 1H, ArH), 7.82 (d, J = 8.2 Hz, 3H, ArH), 7.66 (t, J = 7.5 Hz, 1H, ArH), 6.65
(s, 1H, ArH), 6.61(s, 1H, ArH), 4.09 (s, 3H, OCH3), 3.99 (t, J = 6.6 Hz, 2H, OCH2), 3.85 (d, J =
5.6 Hz, 2H, -NCH2Ar), 3.82 (s, 3H, ArOCH3), 3.76 – 3.63 (m, 4H, -OCH2,-OCH2), 3.59 (s, 2H,-
NCH2Ar), 2.84 (dd, J1 = 10.7, J2 = 4.4 Hz, 4H, -NCH2CH2Ar), 2.51 – 2.44 (m, 6H, -NCH2, -
NCH2, -NCH2), 2.00-1.95 (m, 2H, CH2). 13
C NMR (75 MHz, CDCl3) δ 168.0, 163.8, 155.7,
150.1, 148.0, 147.8, 147.0, 146.6, 146.6, 145.1, 141.2, 137.8, 137.3, 136.8, 131.8, 131.3, 130.3,
130.3, 130.0, 129.4, 128.3, 127.7, 127.5, 127.0, 126.6, 126.4, 122.0, 119.5, 118.9, 116.8, 112.0,
111.5, 67.4, 66.9, 62.6, 56.1, 55.8, 55.5, 53.7, 53.7, 52.5, 51.1, 28.7, 28.5, 26.3. HRMS (EI-MS)
[M+H]+ calcd for C45H45N5O6 752.3443, found 752.3448.
Methyl4-(6-((6-methoxy-7-(2-(piperidin-1-yl)ethoxy)-3,4-dihydroisoquinolin-2(1H)-yl) methyl)-
quinolin-2-yl)-2-(quinoline-2-carbonylamino)benzoate (33f)
Compound 33f was prepared according to the general procedure. The crude product was purified
with flash column chromatography (EtOAc→CHCl3 : MeOH = 20:1) to obtain 33f as pale
yellow solid. Mp. 163°C (decomposition), 1H NMR (600 MHz, CDCl3) δ 13.39 (s, 1H, -CONH),
Chapter 4 | 125
9.81 (d, J = 1.7 Hz, 1H, ArH), 8.43 (d, J = 8.4 Hz, 1H, ArH), 8.39 (d, J = 8.5 Hz, 1H, ArH), 8.37
(d, J = 8.4 Hz, 1H, ArH), 8.29 (d, J = 8.3 Hz, 1H, ArH), 8.26 (d, J = 8.5 Hz, 1H, ArH), 8.19 (d, J
= 8.6 Hz, 1H, ArH), 8.12 (dd, J1 = 8.3, J2=1.7 Hz, 1H, ArH), 8.08 (d, J = 8.6 Hz, 1H, ArH), 7.93
(d, J = 7.9 Hz, 1H, ArH), 7.84 (dtd, J1 = 8.3, J2 = 6.8, J3 = 1.6 Hz, 3H, ArH), 7.69 − 7.66 (m, 1H,
ArH), 6.62 (s, 1H, ArH), 6.54 (s, 1H, ArH). 4.11 (s, 3H, OCH3), 3.87 (s, 2H, -NCH2Ar), 3.82 (s,
3H, ArOCH3), 3.61 (s, 2H, -NCH2Ar), 2.86 (t, J = 5.5Hz, 2H, -NCH2), 2.80 (t, J = 5.6 Hz, 2H, -
NCH2), 2.59 (s, br, 4H, -NCH2, -NCH2), 1.66 − 1.46(m, 6H, CH2CH2CH2). 13
C NMR (151 MHz,
CDCl3) δ 168.0, 163.8, 155.7, 150.2, 148.1, 147.8, 146.7, 145.2, 141.2, 137.7(2C), 137.4, 136.8,
131.8, 131.3, 130.3, 130.2, 130.0(2C), 129.4, 128.4, 127.7, 127.4, 126.9, 126.8(2C), 122.0 ,
119.5, 119.5, 118.9, 116.8, 112.0, 62.6, 57.4, 56.0, 55.8, (2C), 54.7, 52.5(2C), 50.9, 29.7,
28.8(2C), 24.3. HRMS (EI-MS) [M+H]+ calcd for C45H45N5O5 736.3493, found 736.3498.
Methyl4-(6-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)-3,4-dihydroisoquinolin-2(1H)-yl) methyl)-
quinolin-2-yl)-2-(quinoline-2-carbonylamino)benzoate (33g)
Compound 33g was prepared according to the general procedure. The crude product was purified
with flash column chromatography (EtOAc→CHCl3 : MeOH = 20:1) to obtain 33g as pale
yellow solid. Mp. 156°C (decomposition)，1H NMR (600 MHz, CDCl3) δ 13.36 (s, 1H, -
CONH), 9.79 (d, J = 1.6 Hz, 1H, ArH), 8.41 (d, J = 8.4 Hz, 1H, ArH), 8.36 (dd, J1 = 8.2Hz, J2 =
7.8Hz, 2H, ArH), 8.26 (d, J = 8.3 Hz, 1H, ArH), 8.23 (d, J = 8.5 Hz, 1H, ArH), 8.19 (d, J = 8.3
Hz, 1H, ArH), 8.09 (dd, J1 = 8.3, J2 = 1.7 Hz, 1H, ArH), 8.05 (d, J = 8.6 Hz, 1H, ArH), 7.90 (d, J
= 7.6 Hz, 1H, ArH), 7.83 − 7.82 (m, 2H, ArH), 7.81 (d, J = 2.1Hz, 1H, ArH), 7.66 − 7.63 (m, 1H,
ArH), 6.61 (s, 1H, ArH), 6.51 (s, 1H, ArH). 4.09 (s, 3H, OCH3), 3.97 (t, J = 6.7 Hz, 2H, CH2),
3.85 (s, 2H), 3.81 (s, 3H, ArOCH3), 3.59 (s, 2H, -NCH2Ar), 2.85 (t, J = 6.0 Hz, 2H, -NCH2),
2.80 (t, J = 5.4 Hz, 2H, CH2Ar), 2.48 (t, J = 7.2Hz, 2H, -NCH2), 2.41 (s, br, 4H, -NCH2, -NCH2),
2.02 – 1.97 (m, 2H, OCH2), 1.59 – 1.54 (m, 4H, CH2CH2), 1.41 (s, br, 2H, CH2). 13
C NMR (151
MHz, CDCl3) δ 167.9, 163.7, 155.6, 150.0, 148.0, 147.8, 146.6, 146.6, 145.0, 141.1, 137.7,
137.3, 136.7, 131.7, 131.2, 130.2, 130.2, 129.9, 129.3, 128.2, 127.6, 127.4, 126.9, 126.6, 126.4,
122.0, 119.4, 119.4, 118.8, 116.7, 112.0, 111.6, 67.7, 62.6, 56.0, 55.7, 55.7, 54.3(2C), 52.4, 51.0,
28.7, 26.4, 25.6(2C), 24.2. HRMS (EI-MS) [M+H] +
calcd for C46H47N5O5 750.3650, found
750.3659.
Chapter 4 | 126
Methyl 4-(6-((6-methoxy-7-(2-(pyrrolidin-1-yl)ethoxy)-3,4-dihydroisoquinolin-2(1H)-yl) methyl)-
quinolin-2-yl)-2-(quinoline-2-carbonylamino)benzoate (33h)
Compound 33h was prepared according to the general procedure. The crude product was purified
with flash column chromatography (EtOAc→CHCl3 : MeOH = 20:1) to obtain 33h as pale
yellow solid. Mp. 158°C (decomposition), 1H NMR (300 MHz, CDCl3) δ 13.40 (s, 1H, -CONH),
9.81 (d, J = 1.7 Hz, 1H, ArH), 8.44 (d, J = 8.6 Hz, 1H, ArH),8.40 (d, J = 8.6Hz, 1H, ArH),8.38
(d, J = 8.6Hz, 1H, ArH), 8.30 (d, J = 6.4 Hz, 1H, ArH), 8.27 (d, J = 6.6 Hz, 1H, ArH), 8.20 (d, J
= 8.6 Hz, 1H, ArH), 8.11 (dd, J1 = 8.4, J2 = 1.7 Hz, 2H, ArH), 8.08 (d, J = 8.7 Hz, 1H, ArH),
7.93 (d, J = 8.1Hz, ArH), 7.85 (m, 3H, ArH), 6.63 (s, 1H, ArH), 6.57 (s, 1H, ArH), 4.43 (s, br,
2H, ArOCH2), 4.12 (s, 3H, OCH3), 3.90 (d, J = 2.1 Hz, 2H, -NCH2Ar), 3.82 (s, 3H, OCH3), 3.62
(s, 2H, -NCH2Ar), 3.43 (s, 2H, -NCH2), 2.87(s, 2H, CH2Ar), 2.82 (s, 2H, -NCH2), 2.15 (s, br, 4H,
CH2, CH2), 1.34 – 1.20 (m, 4H, CH2CH2). 13
C NMR (75 MHz, CDCl3) δ 168.0, 163.8, 155.7,
150.1, 148.1, 147.8, 146.6, 145.4, 145.1, 141.2, 137.8, 137.2, 136.9, 131.8, 131.3, 130.3, 130.2,
130.0, 129.4, 128.3, 127.8, 127.7, 127.4, 127.1, 126.8, 122.1, 119.5, 119.5, 118.9, 116.8, 112.6,
112.0, 65.9, 62.5, 55.9, 55.6, 54.3(2C), 54.2, 52.6, 50.8, 28.8, 23.3(2C). HRMS (EI-MS) [M+H]+
calcd for C44H43N5O5 738.3286, found 738.3293.
Assay protocol for the determination of ABCB1 and ABCG2 inhibition
Drugs and Chemicals Used for Assays
Topotecan and vinblastine were purchased from Sigma (Munich, Germany), diluted in 70%
ethanol to a concentration of 0.1 mM and stored at 4°C. Hoechst 33342 (Invitrogen, Karlsruhe,
Germany) was dissolved in sterile water at a concentration of 0.8 mM. Calcein-AM (4 mM in
anhydrous DMSO) and pluronic F127 were obtained from Biotium (Hayward, CA, USA).
Fumitremorgin C (FTC; Merck, Darmstadt, Germany) was dissolved in DMSO and diluted to a
concentration of 1 mM. Tariquidar was synthesised in our laboratory according to the
literature[18]
with slight modifications.[19]
The test compounds were dissolved in DMSO at a
concentration of 10 mM. All stock solutions were stored at −20°C. PBS (phosphate buffered
saline) was made of 8.0 g/L NaCl, 1.0 g/L Na2HPO4 · 2H2O, 0.20 g/L KCl, 0.20 g/L KH2PO4 and
0.15 g/L NaH2PO4·H2O and adjusted to pH 7.4. A solution of 4% (m/m) paraformaldeyde (PFA)
in PBS was made by stirring 2 g of PFA per 50 g total solution while heating on a magnetic
Chapter 4 | 127
stirrer for approximately 30 min. If not otherwise stated, chemicals (p.a. quality) were obtained
from Merck (Darmstadt, Germany). Purified water (Milli-Q system, Millipore, Eschborn,
Germany) was used throughout.
Cell lines and Culture Conditions
MCF-7/Topo cells, an ABCG2 overexpressing subclone of MCF-7 cells (ATCC® HTB-22™,
American Type Culture Collection, Rockville, MD, USA), were obtained as described[19]
and
cultured in water-saturated atmosphere (95% air, 5% CO2) at 37°C in 75 cm2 culture flasks from
Nunc (Wiesbaden, Germany) in Eagle’s Minimum Essential Medium (EMEM; Sigma, Munich,
Germany) containing L-glutamine, 2.2 g/L NaHCO3 and 110 mg/L sodium pyruvate
supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany) and 550 nM
topotecan to induce overexpression of the ABCG2 transporter. Human Kb-V1 cells, an ABCB1
overexpressing subclone of Kb cells (ATCC® CCL-17™), were obtained and cultured as
described.[19]
All cells were routinely monitored for mycoplasma contamination by PCR (Venor®
GeM, Minerva Biolabs, Berlin, Germany) and only mycoplasma negative cultures were used.
Modulation of ABCB1 (p-gp): Determination in the Calcein-AM microplate assay
The assay was performed as described.[11]
Modulation of ABCG2: Determination in the Hoechst 33342 microplate assay
MCF-7/Topo cells were seeded into 96-well plates at a density of 20000 cells/well (total volume
100 µL) and allowed to attach to the surface of the microplates overnight in a water-satured
atmosphere (95% air, 5% CO2) at 37°C. The next day, the culture medium was removed, and the
cells were incubated with loading suspension: EMEM (supplemented as described above) and
8 µM Hoechst 33342 in combination with the test compound at increasing concentrations (10 nM
- 100 µM) for 2 h (37°C, 5% CO2). FTC at a final concentration of 10 μM served as reference
compound; under these conditions the response was defined as 100% inhibition of Hoechst
33342 efflux. The supernatants were drained and the cells were fixed for 20 min under light
protection using 100 µL per well of a 4% PFA solution. Finally, MCF-7/Topo cells were washed
twice with 250 µL of PBS per well to remove residual dye. Afterwards, cells were overlaid with
Chapter 4 | 128
100 µL of PBS and the fluorescence intensities were determined using a GENios Pro microplate
reader (TECAN Deutschland GmbH, Crailsheim, Germany). Measurement mode: fluorescence
top; excitation filter: 340/35 nm; emission filter: 485/20 nm; number of reads: 10; integration
time: 40 μs; lag time: 0 μs; mirror selection: automatic; plate definition file: GRE96ft.pdf;
multiple reads per well (circle, 3x3); time between move and flash: 50 ms. On each plate, the
optimal gain was calculated by determination of the fluorescence intensity in the presence of the
reference compound fumitremorgin C. By analogy with the protocol for the calcein-AM assay,
the obtained fluorescence values were normalized with respect to the number of cells per well
(crystal violet staining). All values were corrected by subtracting the fluorescence intensity in
the absence of ABCG2 modulator (DMSO control value), and the maximal response was
referred to the signal caused by 10 μM of the reference compound FTC (100%). IC50 values were
calculated using SIGMA PLOT 11.0, “Four parameter logistic curve” fitting. Errors were
expressed as standard error of the mean (SEM).
Assay protocol for the determination of ABCC1 inhibition
Drugs and chemicals used for assays
Calcein-AM (4 mM in anhydrous DMSO) and pluronic F127 were obtained from Biotium
(Hayward, CA, USA). Bovine serum albumin (BSA) was purchased from Serva (Heidelberg,
Germany. Reversan (Tocris Bioscience, Bristol, UK) was dissolved in DMSO and diluted to a
concentration of 3 mM.
The test compounds were dissolved in DMSO at a concentration of 10 mM if possible,
depending on the solubility of the compounds. All stock solutions were stored at −20°C. Loading
buffer was made of 120 mM NaCl, 5 mM KCl, 2 mM MgCl2∙6H2O, 1.5 mM CaCl2∙2H2O, 25
mM HEPES, 10 mM glucose, pH 7.4.
PBS (phosphate buffered saline) was made of 8.0 g/L NaCl, 1.0 g/L Na2HPO4·2H2O, 0.20 g/L
KCl, 0.20 g/L KH2PO4 and 0.15 g/L NaH2PO4·H2O. The pH value was adjusted to 7.3 − 7.4. A
solution of 4% (m/m) paraformaldeyde (PFA) in PBS was made by stirring 2 g of PFA per 50 g
total solution while heating on a magnetic stirrer for approximately 30 min. If not otherwise
Chapter 4 | 129
stated, chemicals (p.a. quality) were obtained from Merck (Darmstadt, Germany). Purified water
(Milli-Q system, Millipore, Eschborn, Germany) was used throughout.
Cell line and culture conditions
MDCKII-MRP1 cells: MDCKII cells (Madin-Darby Canine Kidney cells, strain II; an epithelial
cell line; ATCC® CRL-2936), transfected with the gene encoding human ABCC1, were a kind
gift from Prof. Dr. P. Borst (Netherland Cancer Institute, Amsterdam, NL). The cells were
cultured in Dulbecco’s Minimum Essential Medium (DMEM; Sigma, Munich, Germany)
supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany), 3.7 g/L of sodium
hydrogen carbonate and 110 mg/L of sodium pyruvate.
Calcein-AM (MRP1) standard protocol
MDCKII-MRP1 cells were seeded into flat-bottomed 96-well plates at a density of 20000-25000
cells per well. On the following day, cells were washed with loading buffer in order to remove
unspecific serum esterases. Afterwards, cells were incubated with loading suspension (loading
buffer, 5 mg/mL BSA, 1.25 μL/mL pluronic F127 (20% in DMSO)) containing 0.5 μM calcein-
AM and the test compound at increasing concentrations (10 nM – 100 µM) for 60 min (37°C /
5% CO2). In general, test compounds were investigated in triplicate, controls in sextuplicate,
respectively. Reversan served as positive control at a final concentration of 30 μM corresponding
to 100% ABCC1 inhibition.
Subsequently, the loading suspension was discarded, and the cells were fixed with 4% PFA
solution in PBS for 20 min. After three washing cycles (loading buffer), fixed cells were overlaid
with loading buffer and relative fluorescence intensities were determined at 535/25 nm at a
GENios Pro microplate reader (TECAN Deutschland GmbH, Crailsheim, Germany) after
excitation at 485/20 nm.
Chapter 4 | 130
TECAN instrument settings: Measurement mode: fluorescence top; number of reads: 10;
integration time: 40 μs; lag time: 0 μs; mirror selection: Dichroic 3 (e.g. Fl.); plate definition file:
GRE96ft.pdf; multiple reads per well (Circle): 3x3; time between move and flash: 100 ms.
The following cell quantification procedure was performed by analogy with the protocol for the
Hoechst 33342 assay. All values were corrected by subtraction of the fluorescence intensity in
the absence of ABCC1 modulators (DMSO control value) and the maximal response was
referred to the signal caused by 30 μM of the reference compound reversan (100%). IC50 values
were calculated using SIGMA PLOT 11.0, “Four parameter logistic curve” fitting. Errors were
expressed as standard error of the mean (SEM).
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Chapter 4 | 132
1H and
13C NMR spectra of selected final compounds
1H and
13C NMR spectra for methyl 4-(6-((6, 7-dimethoxy-3, 4-dihydroisoquinolin-2(1H)-yl)
methyl) quinolin-2-yl)-2-(quinoline-2-carboxamido) benzoate (33a) (600 MHz, CDCl3)
Chapter 4 | 133
1H and
13C NMR spectra for methyl 4-(6-((6-methoxy-7-(2-(2-(2-methoxyethoxy) ethoxy)
ethoxy)-3, 4-dihydroisoquinolin-2(1H)-yl) methyl) quinolin-2-yl)-2-(quinoline-2-carboxamido)
benzoate (33c) (300 MHz, CDCl3)
Chapter 4 | 134
1H and
13C NMR spectra for methyl 4-(6-((6-methoxy-7-(3-morpholinopropoxy)-3, 4-
dihydroisoquinolin-2(1H)-yl) methyl) quinolin-2-yl)-2-(quinoline-2-carboxamido) benzoate (33e)
(300 MHz, CDCl3)
Chapter 5 | 135
This chapter has been published.
Q. Sun, C. J. Yao and B. König. Photochem. Photobiol. Sci, 2015, DOI: 10.1039/C5PP00009B.
Author contributions:
Q.Sun carried out all the photoreactions and wrote the manuscript. C. J. Yao synthesized
compounds 1k-1m given in Table 2.
Chapter 5
Triphenylphosphine mediated photo-rearrangement and methanol addition of
aryl chalcones to 1-propanones
Abstract
Aryl chalcones rearrange and add methanol giving substituted propane-1-ones upon UV-A
irradiation in the presence of PPh3. We propose two possible mechanisms for this photo-
rearrangement. The reaction involves either the formation of a phosphine-carbonyl intermediate,
nucleophilic addition of MeOH and 1, 2 aryl migration or the formation of ylide and carbene
intermediates. Intermediates trapped from the reaction mixture support the first mechanistic
hypothesis.
Keywords
Photo-rearrangement, triphenylphosphine, chalcone, methanol
Chapter 5| 136
Introduction
The photo chemistry of chalcones has always attracted the interest of organic chemists
and in recent years particular the reactions of aryl enones under visible light photoredox
catalysis were studied.[1]
Typical reaction conditions use a ruthenium complex as visible
light absorbing photoredox catalyst and a tertiary amine as sacrificial electron donor to
initiate a photoinduced electron transfer reducing the enone to the corresponding radical
anion, which undergoes e.g. inter- or intramolecular [2 + 2] cycloaddition[1d, 1g]
or a
reductive coupling.[1i]
The photochemistry of aryl ketones in the presence of PPh3 was
studied already more than 40 years ago,[2]
but investigations focused on the
photogeneration of ylides [3]
and Norrish type II reactions.[2d]
Pandey et al. described in
1997 a photocatalytic system for the reductive cyclizations of enones, where DCA were
employed as photoredox catalyst and PPh3 as sacrificial electron donor.[4]
In addition to
its role as electron donor similar to tertiary amines, PPh3 has some unique properties: It is
sterically more hindered; it is no hydrogen atom donor and an efficient quencher of the
carbonyl triplet state (Scheme 1). Therefore the photochemical behavior of α, β-
unsaturated ketones in the presence of PPh3 caused our interest.
Scheme 1. Photolysis of aryl ketones in the presence of PPh3.
We investigated the photoreaction of a variety of chalcone derivatives in the presence of PPh3
applying different solvents, catalysts and light sources. The reaction of chalcone 1a with 10 mol %
of DCA and PPh3 (1 equiv.) in MeOH after 20 h irradiation at 400 nm gave an unexpected
rearrangement and methanol addition product 2a (Scheme 2) instead of the expected cyclization
or Michael addition product. Further studies showed that the reaction proceeds without addition
of a photosensitizer, but not in the dark indicating a direct photochemical process. Similar
rearrangements have been performed using hypervalent iodine [5]
or thallium reagents through
oxidative processes.[6]
However, since PPh3 is not an oxidative reagent, we propose a different
mechanism and developed a convenient experimental procedure for the interesting
rearrangement.
Chapter 5| 137
Scheme 2. Photo-rearrangement and methanol addition of 2-thienyl chalcone 1a.
Results and Discussion
Initially the required amount of PPh3 for the photoreaction was investigated (Table 1). The
desired product was obtained in 84% yield with 0.5 equiv. of PPh3. Decreasing the amount of
PPh3 to 0.25 equiv. does not reduce the product yield, but with catalytic amounts of less than 10
mol% PPh3, the yield of the reaction dropped to 28%. During work 30 to 40% of the PPh3 could
be recycled by column chromatography. Triphenylphosphineoxide was isolated as a byproduct.
The results indicate that PPh3 acts as a catalyst, but decomposes during the reaction yielding
PPh3=O. Using PPh3=O, PHPh2 and DIPEA instead of PPh3 did not yield the desired product 2a,
but the formation of small amounts of [2 + 2] cycloaddition product was observed. Control
experiments without PPh3, without light or under reflux conditions gave no product revealing
that PPh3 and light are essential (Entries 8, 9 and 10). The solvent MeOH was replaced by EtOH,
i-PrOH or CF3CH2OH, but no product formation was detected in these solvents by GC-MS
analysis of the reaction mixture. Besides, the reaction was also carried out with photosensitizers,
e.g., Ru(bpy)3Cl2∙6H2O and Eosin Y, at 450nm and 530nm respectively. The formation of [2 + 2]
cycloaddition product and a reductive coupling product was observed when Ru(bpy)3Cl2∙6H2O
was used in the reaction, while no reaction occurred, when it was irradiated with Eosin Y at
530nm.
Chapter 5| 138
Table 1: Investigation of different reaction conditions for the photo-rearrangement/addition reaction of 1a.
Entry Conditions Yield[%][a][b]
1 PPh3 (1.0 equiv) 56
2 PPh3 (0.5 equiv) 84
3 PPh3 (0.25 equiv) 73
4 PPh3 (0.10 equiv) 28
5 PPh3=O (1.0 equiv) 0
6 PHPh2 (1.0 equiv) 0
7 DIPEA (1.0 equiv) 0
8 no PPh3 0
9 PPh3 (1.0 equiv), no light 0
10 PPh3 (1.0 equiv), no light, reflux 0
[a] Isolated yield. [b] The reactions were carried out in 1.0 mL of CH3OH under N2 atmosphere.
Next, we investigated the substrate scope of aryl chalcones for the photoreaction and the
results are summarized in Table 2. Phenyl and naphthyl chalcones rearrange using the
described reaction conditions. The X-ray structure analysis of compound 2n confirmed its
structure (Figure 1) as assigned from spectroscopic data. Chalcones bearing moderate
electron withdrawing, neutral and electron donating substitutes reacted smoothly affording
the corresponding products in moderate to good yields. Strong electron withdrawing and
donating substitutes like -OCH3, -NO2 and -CN on either aromatic ring inhibit the
rearrangement reaction; products of the [2 + 2] cycloaddition were observed in these cases.
Chapter 5| 139
Table 2: Scope of aryl chalcones in the photo-rearrangement/addition reaction.
Entry Aryl-chalcone R1 R2 product Yield
[%][a],[b]
1 1a 2-thienyl Ph 2a 84
2 1b 2-thienyl 4-F-C6H4 2b 42
3 1c 2-thienyl 4-Br-C6H4 2c 55
4 1d 2-thienyl 4-Cl-C6H4 2d 37
5 1e 2-thienyl 3-Br-C6H4 2e 32
6 1f 2-thienyl 4-Me-C6H4 2f 79
7 1g 2-thienyl 4-MeO-
C6H4
2g trace
8 1h 2-thienyl 4-CN-C6H4 2h -
9 1i Ph Ph 2i 78
10 1j Ph 4-Me-C6H4 2j 60
11 1k 4-MeO-C6H4 4-Br-C6H4 2k -
12 1l 4-MeO-C6H4 Ph 2l trace
13 1m 2-NO2-C6H4 Ph 2m -
14 1n 2-naphthyl Ph 2n 48
[a] Isolated yield. [b] The reactions were carried out in 1.0 mL of CH3OH under N2 atmosphere.
Chapter 5| 140
Figure 1. Structure of compound 2n obtained from the photo-rearrangement/addition reaction of aryl
chalcone 1n, in the solid state.
Several reactions were performed to investigate the mechanism of the photo-
rearrangement/addition reaction. The presence of the persistent radical TEMPO did not
affect the reaction and no radical trapping products were identified indicating the absence of
a radical mechanism. Based on Fox’s earlier mechanistic proposal,[3]
a phosphonium ylide
could be formed through a carbene intermediate upon irradiation. However, our attempts to
trap the phosphonium ylide by reaction with benzaldeyde or the carbene with styrene and
cyclohexene were without success.
Scheme 3. Photo-rearrangement/addition reaction of 1a in CD3OD leads to deuterium incorporation.
Reaction conditions: a) 0.5 equiv PPh3, CD3OD, 20°C, 20 h.
Several reactions were performed to investigate the mechanism of the photo-
rearrangement/addition reaction. Initially, the reaction was performed in deuterated
methanol giving products 1o and 1o’ in a ratio of approximately 5:1 (Scheme 3). The more
Chapter 5| 141
acidic α-hydrogen atom is fully deuterated, while the less reactive β-hydrogen atoms are not
completely exchanged by deuterium.
The presence of the persistent radical 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) did
not affect the reaction and no radical trapping products were identified indicating the
absence of a radical mechanism. On the basis of related reports,[2d, 3, 7]
we propose two
possible mechanisms shown in Scheme 4. The photochemical excitation of the α,β-
unsaturated ketone 1 and ISC gives its triplet state 13, which is captured by PPh3 forming 3
through the intervention of an exciplex. The electronic structure of compound 3 is described
by four resonance structures. In pathway 1, compound 3 is nucleophilic attacked by the
solvent MeOH giving intermediate 4. 1,2-Aryl shift and 1,2-hydrogen shift provide the
product 2 of a rearrangement addition reaction sequence. In pathway 2, 1,2-aryl migration
occurres giving phosphonium ylide 5. It is known that ylides can be photochemically
cleaved to carbenes.[7]
Carbene 6 is formed upon irradiation and quickly trapped by MeOH
to give product 2. Our attempts to identify the intermediate presence of a phosphonium ylide
by reaction with benzaldehyde or of the carbene with styrene, cyclohexene and other
alcohols were without success. We also did not observe any products arising from the
carbene intermediate as described in previous reports.[2d, 3]
Intermediate 3 tends to react via
O-P bond cleavage to the products.[8]
The extendedπsystem of the α,β-unsaturated ketone
may stabilize the carbonyl group. Therefore, the mechanistic hypothesis of pathway 1 may
be more likely, but we cannot rule out a reaction along pathway 2 from our experimental
results.
Chapter 5| 142
Scheme 4. Suggested mechanistic hypothesis of the photo-rearrangement/addition reaction of aryl
chalcones in the presence of PPh3
To demonstrate a synthetic application, we used the photoreaction product 2i for the synthesis of
2-substituted enone 7 (Scheme 5). Functionalized terminal enones are useful compounds in
organic synthesis. They are highly reactive and can undergo conjugate addition reactions with
nucleophiles yielding a variety of bioactive products. The reaction of chalcone 1i with PPh3
under standard photoreaction conditions provided the corresponding product 2i, which was then
further converted into 1,2-diphenylprop-2-en-1-one 7 by heating to 160−170°C with 1%
NaOCH3 in toluene.[9]
Chapter 5| 143
Scheme 5. Synthesis of the 2-substituted terminal enone 7 using the PPh3 mediated photo-
rearrangement/addition reaction of compound 1i and subsequent elimination of methanol. Reaction
conditions: a) 0.5 equiv PPh3, CH3OH, 20°C, 20 h, 78%. b) 1% NaOMe, PhCH3, 160−170°C, 1.5 h, 72%.
Conclusions
In conclusion, we have reported the rearrangement and methanol addition reaction of aryl
chalcones mediated by PPh3 under UV-A irradiation. The reaction proceeds smoothly at
room temperature without sensitizers using 400 nm emitting LEDs. The rearrangement
product can be further converted into 2-substituted terminal enones, which are interesting
molecular structures with potential biologically activity. On the basis of previous reports
and our experiments, we proposed a mechanism involving the triplet state of the arly
chalcone quenched by PPh3. Nucleophilic addition of MeOH to this intermediate, 1,2-aryl
and 1,2-hydrogen atom migration afford diaryl-1-propanones.
Experimental
General Information
1H,
13C NMR spectra were obtained at 298 K using a Bruker AVANCE 300 spectrometer
(operating at 300.13 MHz for 1H and 75.47 MHz for
13C), Bruker AVANCE 400 spectrometer
(operating at 400.13 MHz for 1H and 100.62 MHz for 13C). The spectra were obtained using
chloroform-d (99.8%, Deutero GmbH) and referenced against non-deuterated (1H) / deuterated
(13
C) solvents. The shift values (δ H and δ C) are always given in ppm with J values in Hz. The
melting points were measured using a Stanford Research Systems OptiMelt MPA 100. The
highresolution mass spectra were obtained using a Finnigan MAT SSQ 710A spectrometer at 70
eV (HREIMS, positive and negative mode) or an Agilent 6540 UHD (HRESIMS, positive and
Chapter 5| 144
negative mode). Automated flash chromatography was performed on a Biotage® IsoleraTM
Spektra One device. Silica gel 60 M (40-63 μm, Merck) was used for the flash column
chromatography. The starting materials and reagents were purchased from commercial suppliers
and used without further purification. The solvents were p.a. grade for the reaction mixtures and
industrial grade for the flash column chromatography. Analytical TLC was performed on silica
gel coated alumina plates (MN TLC sheets ALUGRAM® Xtra SIL G/UV254). The visualization
was performed using UV-light (254 and 366 nm). UV–Vis analyses were performed with Varian
Cary 50 UV/Vis spectrophotometer and Agilent 8453 UV-Vis Spectrometer. For UV
measurements 10 mm Hellma fluorescence quartz cuvettes (117.100F-QS) with a screw cap with
PTFE-coated silicon septum were used. Chalcone 1i was purchased from sigma-aldrich.
Irradiation Source:
Philips LUXEON® Rebel (purple, max = 400 ± 10 nm, 1000 mA, 1.2 W)
General procedure for preparation of α,β-unsaturated ketones 1a-1h and 1j-1n.
To a stirred solution of acetophenone (10 mmol) in methanol (5 mL) was added dropwise a
solution of sodium hydroxide (13 mmol) in methanol (10 mL). Fifteen minutes later, the
resulting mixture was further treated with substituted benzaldehydes (10 mmol) and stirred at
room temperature. When the reaction was complete (disappearance of acetophenone, monitored
by TLC), 40 mL water was added. The solid products were filtered off, washed with water (3 ×
25 mL), cold methanol (3 × 25 mL) and dried to give corresponding α,β-unsaturated ketones.[10]
(E)-3-Phenyl-1-(thiophen-2-yl)prop-2-en-1-one (1a)
Pale yellow powder. 1H NMR (300 MHz, CDCl3) δ 7.91 – 7.81 (m, 2H), 7.69 (dd, J = 4.9, 0.9
Hz, 1H), 7.67 -7.67 (m, 2H), 7.47 – 7.39 (m, 4H), 7.20 (dd, J = 4.9, 3.9 Hz, 1H). The
spectroscopy is in accordance with literature.[11]
(E)-3-(4-Fluorophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (1b)
Chapter 5| 145
Pale yellow powder. 1H NMR (300 MHz, CDCl3) δ 7.89 – 7.77 (m, 2H), 7.70 (dd, J = 4.9, 1.0
Hz, 1H), 7.67 – 7.61 (m, 2H), 7.35 (d, J = 15.6 Hz, 1H), 7.23 – 7.16 (m, 1H), 7.16 – 7.07 (m,
2H). The spectroscopy is in accordance with literature.[12]
(E)-3-(4-Bromophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (1c)
Pale yellow powder. 1H NMR (300 MHz, CDCl3) δ 7.87 (dd, J = 3.8, 1.0 Hz, 1H), 7.78 (d, J =
15.6 Hz, 1H), 7.70 (dd, J = 4.9, 1.0 Hz, 1H), 7.59 – 7.48 (m, 4H), 7.45 – 7.35 (m, 1H), 7.20 (dd,
J = 4.9, 3.9 Hz, 1H). The spectroscopy is in accordance with literature.[13]
(E)-3-(4-Chlorophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (1d)
White powder. 1H NMR (300 MHz, CDCl3) δ 7.87 (dd, J = 3.8, 0.9 Hz, 1H), 7.80 (d, J = 15.6 Hz,
1H), 7.70 (dt, J = 9.3, 4.7 Hz, 1H), 7.59 (dd, J = 8.8, 2.2 Hz, 2H), 7.44 – 7.34 (m, 3H), 7.20 (dd,
J = 4.9, 3.9 Hz, 1H). The spectroscopy is in accordance with literature.[14]
(E)-3-(3-Bromophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (1e)
White powder. 1H NMR (300 MHz, CDCl3) δ 7.89 (dd, J = 3.8, 1.0 Hz, 1H), 7.78 (dd, J = 11.1,
8.9 Hz, 2H), 7.71 (dd, J = 4.9, 1.0 Hz, 1H), 7.58 – 7.51 (m, 2H), 7.41 (d, J = 15.5 Hz, 1H), 7.29
(dd, J = 13.2, 5.3 Hz, 1H), 7.21 (dd, J = 4.9, 3.9 Hz, 1H). The spectroscopy is in accordance with
literature.[12]
(E)-1-(Thiophen-2-yl)-3-(p-tolyl)prop-2-en-1-one (1f)
White powder. 1H NMR (300 MHz, CDCl3) δ 7.89 – 7.79 (m, 2H), 7.68 (dd, J = 4.9, 1.0 Hz, 1H),
7.55 (d, J = 8.1 Hz, 2H), 7.43 – 7.34 (m, 1H), 7.23 (d, J = 8.0 Hz, 2H), 7.19 (dd, J = 4.9, 3.8 Hz,
1H), 2.40 (s, 3H). The spectroscopy is in accordance with literature.[12]
(E)-3-(4-Methoxyphenyl)-1-(thiophen-2-yl)prop-2-en-1-one (1g)
Chapter 5| 146
White powder.
1H NMR (300 MHz, CDCl3) δ 7.92 – 7.75 (m, 2H), 7.67 (dd, J = 4.9, 1.0 Hz,
1H), 7.64 – 7.57 (m, 2H), 7.37 – 7.28 (m, 1H), 7.18 (dd, J = 4.9, 3.8 Hz, 1H), 6.99 – 6.90 (m,
2H). The spectroscopy is in accordance with literature.[12]
(E)-4-(3-Oxo-3-(thiophen-2-yl)prop-1-en-1-yl)benzonitrile (1h)
White powder. 1H NMR (300 MHz, CDCl3) δ 7.89 (dd, J = 3.8, 1.0 Hz, 1H), 7.82 (d, J = 15.6 Hz,
1H), 7.76 – 7.69 (m, 5H), 7.48 (d, J = 15.6 Hz, 1H), 7.22 (dd, J = 4.9, 3.9 Hz, 1H). The
spectroscopy is in accordance with literature.[15]
(E)-1-Phenyl-3-(p-tolyl)prop-2-en-1-one (1j)
White powder. 1H NMR (300 MHz, CDCl3) δ 8.01 (dd, J = 5.3, 3.3 Hz, 2H), 7.80 (d, J = 15.7 Hz,
1H), 7.62 – 7.45 (m, 6H), 7.23 (d, J = 8.0 Hz, 2H). The spectroscopy is in accordance with
literature.[16]
(E)-3-(4-Bromophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (1k)
White powder. 1H NMR (300 MHz, CDCl3) δ 8.11 – 7.95 (m, 2H), 7.73 (d, J = 15.7 Hz, 1H),
7.59 – 7.45 (m, 5H), 7.04 – 6.93 (m, 2H), 3.90 (s, 3H). The spectroscopy is in accordance with
literature.[17]
(E)-1-(4-Methoxyphenyl)-3-phenylprop-2-en-1-one (1l)
White powder. 1H NMR (300 MHz, CDCl3) δ 8.10 – 8.00 (m, 2H), 7.87 – 7.74 (m, 1H), 7.70 –
7.61 (m, 2H), 7.60 – 7.50 (m, 1H), 7.43-7.41 (m, 3H), 7.04 – 6.94 (m, 2H), 3.89 (s, 3H). The
spectroscopy is in accordance with literature.[18]
(E)-1-(2-Nitrophenyl)-3-phenylprop-2-en-1-one (1m)
Chapter 5| 147
White powder. 1
H NMR (300 MHz, CDCl3) δ 8.21 – 8.18 (dd, J = 0.1, 8.2 Hz,1H), 7.81 – 7.75
(m, 1H), 7.70 – 7.64 (m, 1H), 7.54 – 7.50 (m, 3H), 7.41 – 7.36 (m, 3H), 7.26 (d, J = 16.3 Hz, 1H),
7.02 (d, J = 16.3 Hz, 1H). The spectroscopy is in accordance with literature.[19]
(E)-1-(Naphthalen-2-yl)-3-phenylprop-2-en-1-one (1n)
White powder. 1
H NMR (300 MHz, CDCl3) δ 8.55 (s, 1H), 8.11 (dd, J = 8.6, 1.7 Hz, 1H), 8.04 –
7.98 (m, 1H), 7.98 – 7.85 (m, 3H), 7.75 – 7.67 (m, 3H), 7.60 (pd, J = 6.9, 1.5 Hz, 2H), 7.48 –
7.42 (m, 3H). The spectroscopy is in accordance with literature.[20]
General procedure for the photo-rearrangement/addition reaction.
In a 5 mL snap vial equipped with magnetic stirring bar the PPh3 (0.5 equiv, 0.125mmol) and
aryl chalcone derivatives (1.0 equiv, 0.25mmol) were added in 1 mL of CH3OH, and the
resulting reaction mixture was degassed by three “pump-freeze-thaw” cycles via a syringe needle.
The vial was irradiated through the vial’s plane bottom side using 400 nm purple LEDs with
cooling device maintaining a temperature around 20°C. After 20h of irradiation, the solvent was
removed and purified by flash column chromatography using petrol ether (50−70°C)/ethyl
acetate (99:1 to 99:5) as eluent.
3-Methoxy-2-phenyl-1-(thiophen-2-yl)propan-1-one (2a)
Colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.75 (dd, J = 3.8, 1.1 Hz, 1H), 7.61 – 7.57 (m, 1H),
7.39 – 7.34 (m, 2H), 7.32 (ddd, J = 7.6, 4.5, 1.2 Hz, 2H), 7.06 (dd, J = 4.9, 3.9 Hz, 1H), 4.71 (dd,
J = 8.9, 5.3 Hz, 1H), 4.18 (t, J = 9.0 Hz, 1H), 3.64 (dd, J = 9.1, 5.3 Hz, 1H), 3.36 (s, 3H). 13
C
NMR (101 MHz, CDCl3) δ 191.1, 144.1, 136.3, 134.0, 132.7, 129.0, 128.3, 128.1, 127.7, 74.4,
59.2, 55.3. HRMS (EI-MS) calcd for C14H14O2S [M+H]+ 247.0787 found 247.0788.
2-(4-Fluorophenyl)-3-methoxy-1-(thiophen-2-yl)propan-1-one (2b)
Chapter 5| 148
Colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.74 (dd, J = 3.8, 1.1 Hz, 1H), 7.63 – 7.59 (m, 1H),
7.37 – 7.30 (m, 2H), 7.07 (dt, J = 7.3, 3.6 Hz, 1H), 7.04 – 6.96 (m, 2H), 4.73 – 4.66 (m, 1H),
4.13 (t, J = 8.9 Hz, 1H), 3.62 (dd, J = 9.1, 5.5 Hz, 1H), 3.35 (s, 3H). 13
C NMR (75 MHz, CDCl3)
δ 191.1, 143.8, 134.3, 132.7, 129.9, 129.8, 128.2, 116.1, 115.8, 74.3, 59.2, 54.3. HRMS (EI-MS)
calcd for C14H13FO2S [M+H]+ 265.0693 found 265.0696.
2-(4-Bromophenyl)-3-methoxy-1-(thiophen-2-yl)propan-1-one (2c)
Colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.72 (dd, J = 3.8, 1.1 Hz, 1H), 7.60 (dd, J = 4.9, 1.1
Hz, 1H), 7.46 – 7.40 (m, 2H), 7.27 – 7.22 (m, 2H), 7.06 (dd, J = 4.9, 3.9 Hz, 1H), 4.67 (dd, J =
8.5, 5.7 Hz, 1H), 4.20 – 4.03 (m, 1H), 3.62 (dd, J = 9.1, 5.7 Hz, 1H), 3.34 (s, 3H).13
C NMR (75
MHz, CDCl3) δ 190.7, 143.7, 135.4, 134.5, 132.8, 132.1, 130.0, 128.3, 121.8, 74.1, 59.3, 54.5.
HRMS (EI-MS) calcd for C14H13BrO2S [M+H]+ 324.9892 found 324.9894.
2-(4-Chlorophenyl)-3-methoxy-1-(thiophen-2-yl)propan-1-one (2d)
Colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.77 – 7.69 (m, 1H), 7.65 – 7.54 (m, 1H), 7.29 (d, J
= 2.1 Hz, 3H), 7.12 – 6.98 (m, 1H), 4.68 (dd, J = 9.1, 5.7 Hz, 1H), 4.19 – 4.03 (m, 1H), 3.62 (dd,
J = 9.1, 5.7 Hz, 1H), 3.34 (s, 3H). 13
C NMR (75 MHz, CDCl3) δ 190.8, 143.7, 134.8, 134.4,
133.7, 132.8, 129.6, 129.2, 128.3, 74.2, 59.3, 54.5. HRMS (EI-MS) calcd for C14H13ClO2S
[M+H]+ calcd for 281.0398 found 281.0398.
2-(3-Bromophenyl)-3-methoxy-1-(thiophen-2-yl)propan-1-one (2e)
Colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.74 (dd, J = 3.8, 1.0 Hz, 1H), 7.62 (dd, J = 4.9, 1.0
Hz, 1H), 7.53 (dd, J = 6.4, 4.7 Hz, 1H), 7.41 – 7.34 (m, 1H), 7.34 – 7.28 (m, 1H), 7.23 – 7.16 (m,
1H), 7.11 – 7.04 (m, 1H), 4.67 (dd, J = 8.6, 5.6 Hz, 1H), 4.17 – 4.08 (m, 1H), 3.63 (dd, J = 9.1,
5.6 Hz, 1H), 3.34 (s, 3H). 13
C NMR (75 MHz, CDCl3) δ 190.5, 143.8, 138.5, 134.6, 132.9, 131.3,
130.9, 130.5, 128.3, 127.0, 123.0, 74.2, 59.3, 54.7. HRMS (EI-MS) calcd for C14H13BrO2S
[M+H]+ calcd for 324.9892 found 324.9891.
3-Methoxy-1-(thiophen-2-yl)-2-(p-tolyl)propan-1-one (2f)
Chapter 5| 149
Colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.75 (dd, J = 3.8, 0.9 Hz, 1H), 7.54 (dd, J = 4.9, 0.9
Hz, 1H), 7.28 (s, 1H), 7.12 (d, J = 8.0 Hz, 2H), 7.03 (dd, J = 4.9, 3.9 Hz, 1H), 4.71 (dd, J = 8.9,
5.3 Hz, 1H), 4.22 – 4.13 (m, 1H), 3.62 (dd, J = 9.1, 5.3 Hz, 1H), 3.35 (s, 3H), 2.29 (s, 3H). 13
C
NMR (75 MHz, CDCl3) δ 191.3, 144.1, 137.4, 134.0, 133.3, 132.7, 129.7, 128.2, 74.4, 59.2, 54.8,
21.1. HRMS (EI-MS) calcd for C15H16O2S [M+H]+ 261.0944 found 261.0946.
3-Methoxy-1,2-diphenylpropan-1-one (2i)
Colorless oil. 1H NMR (300 MHz, CDCl3) δ 8.02 – 7.95 (m, 2H), 7.50 – 7.45 (m, 1H), 7.43 –
7.37 (m, 2H), 7.32 (dt, J = 8.7, 1.7 Hz, 4H), 7.25 – 7.21 (m, 1H), 4.90 (dd, J = 8.7, 5.3 Hz, 1H),
4.25 – 4.14 (m, 1H), 3.65 (dd, J = 9.1, 5.3 Hz, 1H), 3.36 (s, 3H). 13
C NMR (75 MHz, CDCl3) δ
198.3, 136.7, 136.3, 133.1, 129.0, 128.8, 128.6, 128.4, 127.6, 74.7, 59.2, 53.8. HRMS (EI-MS)
calcd for C16H16O2 [M+H]+
calcd for 241.1223 found 241.1228.
3-Methoxy-1-phenyl-2-(p-tolyl)propan-1-one (2j)
Colorless oil. 1H NMR (300 MHz, CDCl3) δ 8.04 – 7.94 (m, 2H), 7.49 – 7.45 (m, 1H), 7.41-7.29
(m, 2H), 7.23 (d, J = 8.1 Hz, 2H), 7.12 (d, J = 7.9 Hz, 2H), 4.87 (dd, J = 8.7, 5.3 Hz, 1H), 4.18 (t,
J = 8.9 Hz, 1H), 3.63 (dt, J = 11.1, 5.5 Hz, 1H), 3.36 (s, 3H), 2.29 (m, 3H). 13
C NMR (75 MHz,
CDCl3) δ 198.4, 137.3, 136.7, 133.3, 133.0, 129.8, 128.8, 128.5, 128.2, 74.7, 59.2, 53.4, 21.1.
HRMS (EI-MS) calcd for C17H18O2 [M+H]+ 255.1380, found 255.1377.
3-Methoxy-1-(naphthalen-2-yl)-2-phenylpropan-1-one (2n)
Colorless solid. 1H NMR (300 MHz, CDCl3) δ 8.51 (s, 1H), 8.04 (dd, J = 8.7, 1.8 Hz, 1H), 7.92
(d, J = 7.9 Hz, 1H), 7.82 (dd, J = 12.2, 6.8 Hz, 2H), 7.54 (ddd, J = 9.2, 5.1, 1.4 Hz, 2H), 7.41-
7.38 (m, 2H), 7.34 – 7.28 (m, 2H), 7.25 – 7.21 (m, 1H), 5.06 (dd, J = 8.7, 5.3 Hz, 1H), 4.25 (t, J
= 8.9 Hz, 1H), 3.71 (dd, J = 9.1, 5.3 Hz, 1H), 3.38 (s, 3H).13
C NMR (75 MHz, CDCl3) δ 198.3,
136.5, 135.5, 134.1, 132.4, 130.6, 129.7, 129.1, 128.5, 128.4, 127.7, 127.6, 126.7, 124.4, 74.8,
59.2, 53.8. HRMS (EI-MS) calcd for C20H18O2 [M+H]+ 291.1380 found 291.1381.
Chapter 5| 150
1,2-Diphenylprop-2-en-1-one (7)
White solid. 1H NMR (300 MHz, CDCl3) δ 7.95 – 7.86 (m, 2H), 7.60 – 7.32 (m, 8H), 6.08 (s,
1H), 5.65 (s, 1H). The NMR spectra is in accordance with literature.[21]
1H NMR data for [2 + 2] cycloaddition products
(3,4-Diphenylcyclobutane-1, 2-diyl)bis (thiophen-2-yl methanone).
Pale yellow solid. 1H NMR (300 MHz, CDCl3) δ 7.61 (dd, J = 4.9, 1.0 Hz, 2H), 7.43 (dd, J = 3.8,
1.0 Hz, 2H), 7.32 (d, J = 4.4 Hz, 4H), 6.96 (dd, J = 4.9, 3.9 Hz, 1H), 4.49 – 4.37 (m, 2H), 4.05 –
3.92 (m,2). The spectroscopy is in accordance with literature.[22]
(3,4-Diphenylcyclobutane-1,2-diyl)bis((4-methoxyphenyl)methanone).
White solid. 1H NMR (300 MHz, CDCl3) δ 7.85 – 7.77 (m, 4H), 7.31 – 7.29 (m, 8H), 7.25 – 7.21
(m, 2H), 6.82 – 6.74 (m, 4H), 4.59 – 4.49 (m, 2H), 4.02 – 3.93 (m, 2H), 3.79 (s, 6H). The
spectroscopy is in accordance with literature.[23]
UV spectra of chalcone 2a and PPh3 before and after irradiation
The absorbance in the range of 390-410 nm is very weak. Molar absorptivity at 390 nm was in
the range of 100-500 cm-1
. After 20h of irradiation, the reaction mixture turnes to light yellow
color and showed stronger absorption at 390 nm. The peak of chalcone 2a at 324 nm
dramatically decreases demonstrating the consumption of chalcone 2a.
UV–visible spectra of the compounds were determined in MeOH solution (conc. 2×10-5
M).
Chapter 5| 151
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Chapter 5| 152
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Chapter 5| 153
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Chapter 5| 154
1H and
13C NMR spectra of selected compounds
1H and
13C NMR spectra for 2-(4-fluorophenyl)-3-methoxy-1-(thiophen-2-yl)propan-1-one (2b)
(300MHz, CDCl3)
Chapter 5| 155
1H and
13C NMR spectra for 2-(4-bromophenyl)-3-methoxy-1-(thiophen-2-yl)propan-1-one (2c)
(300MHz, CDCl3)
Chapter 5| 156
1H and
13C NMR spectra for 3-methoxy-1-(naphthalen-2-yl)-2-phenylpropan-1-one (2n)
(300MHz, CDCl3)
Chapter 5| 157
Abbreviation
ABCB1
ATP-binding cassette
sub-family B member 1
MS Mass spectrometry
ABCG2 ATP-binding cassette
sub-family G member 2 equiv Equivalent
ABCC1 ATP-binding cassette,
sub-family C member 1 ES Electrospray
CDCl3 Deuterated chloroform ESI Electrospray
ionization
DCM Dichloromethane Et2O Diethyl ether
DMF Dimethylformamide EtOAc Ethyl acetate
DMSO Dimethyl sulfoxide EtOH Ethanol
DMSO-d6 Deuterated dimethyl
sulfoxide eV Electron volts
GC Gas chromatography MeOD Deuterated methanol
HR-MS High resolution mass
spectrometry MeOH Methanol
ISC Inter system crossing MHz Mega hertz
min Minute Mp Melting point
mL Milli liter NMR Nuclear magnetic
resonance
mmol Milli mole PE petroleum ether
ppm Parts per million SCE Saturated calomel
electrode
TEMPO
(2,2,6,6-Tetramethyl-
piperidin-1-yl)oxyl
TLC Thin layer
chromatography
TMS Tetramethylsilane UV Ultra violet
PEG Polyethylene glycol DIPEA Diisopropylethylamin
conc. concentrated Pd/C Palladium on charcoal
Chapter 5| 158
r.t Room temperature THF Tetrahydrofurane
SEM Standard error of the
mean TFA Trifluroacetic acid
Chapter 5| 159
Summary
The first part of the thesis (chapter 1 and 2) deals with natural or natural-like phenols with anti-
angiogenic property. Chapter 1 reviews recent reports of anti-angiogenic natural phenolic
compounds specifically addressing their chemistry, synthesis and possible structure
modifications. Thirteen representatives of eight different natural or natural-like phenolic
subclasses with significant anti-angiogenic activity are presented. Whenever available, structure-
activity relationship is also discussed.
In chapter 2, we describe a synthetic approach for natural and natural-like acylphloroglucinols
with anti-proliferative, anti-oxidative and tube-formation inhibitory activity. Two series of
mono- and bicyclic acylphloroglucinols derived from secondary metabolites in the genus
Hypericum (Hypericaceae) were synthesised and tested in vitro for anti-proliferative and tube-
formation inhibitory activity in human microvascular endothelial cells (HMEC-1). In addition,
their anti-oxidative activity was determined via an ORAC-assay. Our experiments show simpler
acylphloroglucinols containing simpler substitution patterns than hyperforin can show good anti-
proliferative effects and remarkable tube formation inhibition.
The second part of the thesis (chapter 3 and 4) reports the synthesis and bioactive evaluation of
compounds aiming to selectively inhibit ABCC1 and ABCG2 transporter, respectively. In
chapter 3, a series of flavonoids were synthesised and characterized in cellular assays for
modulation of the ABC transporters-ABCC1 (MDCKII-MRP1 cells), ABCB1 (Kb-V1 cells) and
ABCG2 (MCF-7/Topo cells). The most potent ABCC1 modulators identified among these
flavonoid-type compounds were comparable to reversan regarding their potency, but superior in
terms of selectivity over ABCB1 and ABCG2.
In chapter 4, we report synthesis of quinoline analogues targeting breast cancer resistance protein
derived from tariquidar. All tested compounds show weak or no inhibitory activity over ABCB1
and ABCC1 transporter but good inhibitory activity towards ABCG2. However, compared with
our reference indole compounds, the replacement of anilide core by quinoline moiety gave less
potent compounds. The introduction of amine groups on the tetrahydroisoquinoline moiety can
increase the water solubility to some extent, but the poor water solubility is still the main
problem for this series of compounds.
Chapter 5| 160
In chapter 5, photo-rearrangement and methanol addition of aryl chalcones to 1-propanones in
the presence of triphenylphosphine is reported. We propose two possible mechanistic hypotheses
for the rearrangement/addition reaction. To further demonstrate the applicability of the reaction,
we applied it to synthesis of 2-substituted terminal enones.
Chapter 5| 161
Zusammenfassung
Der erste Teil der vorliegenden Arbeit (Kapitel 1 und 2) beschäftigt sich mit natürlichen oder
naturstoffverwandten Phenolen mit antiangiogenetischen Eigenschaften. Kapitel 1 gibt einen
Überblick über phenolgruppenenthaltende Naturstoffe mit Angiogenese hemmender Wirkung,
dabei wird auf ihre Chemie, die Synthese sowie mögliche strukturelle Modifikationen
eingegangen. Dreizehn Vertreter von acht verschiedenen Unterklassen natürlicher oder von
natürlichen Strukturen abgeleitete Phenole mit hoher antiangiogenetischer Aktivität werden
vorgestellt und falls möglich anhand ihrer Struktur-Wirkungsbeziehung diskutiert.
In Kapitel 2 beschreiben wir eine Syntheseroute für natürliche und naturstoffverwandte
Acylphloroglucine mit antiproliferativer, antioxidativer und „Tube Formation“ hemmender
Aktivität. Zwei Serien von natürlichen und naturverwandten mono und bizyklischen
Acylphloroglucinen, abgeleitet von Sekundärmetaboliten der Gattung Hypericum (Hypericaceae),
wurden synthetisiert und in vitro auf ihre antiproliferative Wirkung und ihre
Angiogenesehemmung (Tube Formation Assay) in humanen mikrovaskulären Endothelzellen
(HMEC-1) getestet. Zusätzlich wurde ihre antioxidative Wirkung mittels eines ORAC Assays
bestimmt. Unsere Experimente zeigen, dass einfachere Acylphloroglucine mit einfacherem
Substitutionsmuster als Hyperforin gute antiproliferative Wirkung und bemerkenswerte
Angiogenesehemmung zeigen können.
Der zweite Teil der Arbeit (Kapitel 3 und 4) beschäftigt sich mit der Synthese von Wirkstoffen
zur selektiven Inhibierung des ABCC1 beziehungsweise ABCG2 Transporters, sowie der
Bewertung ihrer biologischen Aktivität. In Kapitel 3 wird die Synthese einer Reihe von
Flavonoiden dargestellt und mittels zellbasierter Assays auf ihre Wirkung auf die ABC
Transporter ABCC1 (MDCKII-MRP1 Zellen), ABCB1 (Kb-V1 Zellen and ABCG2 (MCF-
7/Topo Zellen) untersucht. Die wirksamsten ABCC1 Inhibitoren der Serie zeigen eine
vergleichbare Aktivität wie Reversan, sind diesem jedoch in Hinblick auf die Selektivität
gegenüber der verwandet Transporter ABCB1 und ABCG2 überlegen.
In Kapitel 4 wird die Synthese eines von Tariquidar abgeleiteten Chinolinderivates zur
Inhibierung des Breast Cancer Resistance Proteins beschrieben. Sämtliche untersuchte
Substanzen zeigten keine oder nur sehr schwache Hemmung der ABCB1 und ABCC1
Transporter, besaßen jedoch eine hohe Aktivität gegenüber ABCG2. Allerding zeigte sich im
Chapter 5| 162
Vergleich zu den Indolverbindungen, dass der Austausch der Anilideinheit durch ein Chinolin zu
einer verringerten Aktivität der Verbindungen führte. Durch Einführen einer Aminogruppe am
Tetrahydroisochinolin kann die Wasserlöslichkeit der Verbindung geringfügig verbessert werden,
jedoch bleibt die geringe Wasserlöslichkeit dieser Substanzklasse nach wie vor problematisch.
Kapitel 5 beschreibt die photochemische Umlagerung von Arylchalkonen zu 1-Propanonen in
der Gegenwart von Triphenylphosphin und unter Angriff von Methanol. Für den Mechanismus
dieser Umlagerungs-Additions-Reaktion werden zwei unterschiedliche Hypothesen aufgestellt
und erläutert. Um den synthetischen Nutzen dieser neu entdeckten Reaktion aufzuzeigen, haben
wir sie auf die Darstellung eines 2-substituierten Enones angewendet.
Chapter 5| 163
Curriculum Vitae
Personal data
Qiu Sun
Date of birth: September.29th.1985
Nationality: Chinese
Languages: English, German (A1), Chinese (mother tongue)
E-mail: [email protected]
University education
10/2011-03/2015
Ph.D. in Medicinal Chemistry
Institute of Organic Chemistry, University of Regensburg, Germany
Advisor: Prof.Dr.Burkhard Koenig
Research area:
Syntheses of natural-like acylphloroglucinols for pharmacological testing
Syntheses of ABC modulators
PPh3-mediated photo rearrangement of chalcone analogues
09/2008-07/2011
Master of Science in Medicinal Chemistry
West China School of Pharmacy, Sichuan University, China
Chapter 5| 164
Advisor: Prof.Dr.Taiping Hou
Master thesis:
Synthesis, bioactive and molecular docking study on neonicotinoid analogues
Iron trichloride-assisted selective synthesis of vascular protective agent probucol
monosuccinate
09/2004-07/2008
Bachlor of Science in Pharmacy
West China School of Pharmacy, Sichuan University, China
Advisor: Prof.Dr.Taiping Hou
Bachlor thesis:
Anti-fungal study on metabolites isolated from Ligularia Virgaurea
Skills
Detailed knowledge of spectroscopical analytics in theory and practice (NMR, IR, UV-Vis
and MS), chromatographic methods and cyclic voltammetry
Good knowledge of chemistry software, MS Office and Endnote
Teaching experience
Supervised one undergraduate and four master students during their research projects at the
University of Regensburg
Assisted to supervise master students lab course in 2012
Publication
Chapter 5| 165
Qiu Sun, Sebastian Schmidt, Martina Tremmel, Jörg Heilmann, Burkhard König. Synthesis
of Natural-like Acylphloroglucinols with Anti-proliferative, Anti-oxidative and Tube-
formation Inhibitory Activity, Eur. J. Med. Chem, 2014, 85, 621-628.
Qiu Sun, Jörg Heilmann, Burkhard König. Natural phenolic metabolites with anti-
angiogenic properties – a review from the chemical point of view. Beilstein J. Org. Chem,
2015, 11, 249-264.
Qiu Sun, Chang-jiang Yao, Burkhard König. Triphenylphosphine mediated photo-
rearrangement and methanol addition of aryl chalcones to 1-propanones. Photochem.
Photobiol. Sci., 2015. DOI: 10.1039/C5PP00009B
Jose Obreque-Balboa, Qiu Sun, Bernhardt Günther, Burkhard König and Armin
Buschauer.Synthesis and biological evaluation of flavonoid derivatives as novel and highly
selective ABCC1 modulators. (joint first author, manuscript in preparation)
Chang-jiang Yao, Qiu Sun, Rastogi Namrata, Burkhard König. Intermolecular Formyloxy-
Arylation of Alkenes by Photoredox Meerwein Reaction. ACS Catal. 2015.
DOI: 10.1021/acscatal.5b00314.
Stefanie Bauer, Cristian Ochoa-Puentes, Qiu Sun, Manuel Bause, Günther Bernhardt,
Burkhard König, and Armin Buschauer. Quinoline Carboxamide-Type ABCG2 Modulators:
Indole and Quinoline Moieties as Anilide Replacements. ChemMedChem, 2013, 8, 1-6.
Zhi-yi Yu, Guan-ying Shi, Qiu Sun, Tai-ping Hou, Design, Synthesis and in vitro
Antibacterial/antifungal Evaluation of Novel 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7(1-
piperazinyl)quinoline-3-carboxylic Acid Derivatives, Eur . J. Med. Chem, 2009, 44, 4726-
4733.
Tai-ping Hou, Yun Teng, Qiu Sun, Zhi-yi Yu. A New Fungitoxic Metabolite from Spiraea
alpina Pall. Fitoterapia, 2009, 80, 237-240.
Conference
6th Summer School Medicinal Chemistry, University of Regensburg, September 26-28,
2012
Advances in Drug Discovery – Chemistry and Biology, Prague, Czech Republic, September
1-5, 2014
Chapter 5| 166
7th Summer School Medicinal Chemistry, University of Regensburg, September 17-19,
2014
Chapter 5| 167
Acknowledgement
I would like to express my sincere gratitude
to my research supervisor Prof. Dr. Burkhard König for the freedom to explore projects, the
guidance and assistance when my steps were stagnant ,
to Prof. Dr. Jörg Heilmann, Prof. Dr. Armin. Buschauer and Prof. Dr. Günther Bernhardt for the
excellent cooperation and valuable ideas in the medchem projects,
to all the persons from the pharmacy department which were involved in the pharmacological
part of this work, especially Dr. Sebastian Schmidt, Dr. Stefanie Bauer and José Esteban
Obreque-Balboa for pharmacological testing and explanations,
to Dr. Rudi Vasold and Simone Strauß for GC, Ernst Lautenschlager for his help in all technical
questions, Susanne Schulze for ordering the chemicals, Regina Hoheisel for CV measurements,
to all members of the König-group for their help, ideas and support, especially my lab mates
Thea, Simone, Durga, Manuel, Supratim, Tamal, Daniel,
to Olga Schitkow, Michael Dumin, Mengya Chen, ,Shiwen Xue and Anja Schlicht for their
motivated work during their internships,
to all my friends especially Qingqing Xie, Xueke She, Jianfei Wan, Mengya Chen, Changjiang
Yao, Guozheng Huang, Sookyoung Kang and my German mother Margit for making my stay in
Regensburg wonderful and exciting,
to Wenbo for his encouragement and understanding,
to my parents and my grandfather who support and accompany with me any time through all of
my study.